Polymeric micelles for drug delivery

ABSTRACT

The present invention relates to the field of polymer chemistry and more particularly to multiblock copolymers and micelles comprising the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to United States provisionalapplications Ser. No. 60/667,260, filed Apr. 1, 2005, and 60/741,780,filed Dec. 1, 2005, the entirety of each of which is hereby incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of polymer chemistry and moreparticularly to micelles and uses thereof.

BACKGROUND OF THE INVENTION

The development of new therapeutic agents has dramatically improved thequality of life and survival rate of patients suffering from a varietyof disorders. However, drug delivery innovations are needed to improvethe success rate of these treatments. Specifically, delivery systems arestill needed which effectively minimize premature excretion and/ormetabolism of therapeutic agents and deliver these agents specificallyto diseased cells thereby reducing their toxicity to healthy cells.

Rationally-designed, nanoscopic drug carriers, or “nanovectors,” offer apromising approach to achieving these goals due to their inherentability to overcome many biological barriers. Moreover, theirmulti-functionality permits the incorporation of cell-targeting groups,diagnostic agents, and a multitude of drugs in a single delivery system.Polymer micelles, formed by the molecular assembly of functional,amphiphilic block copolymers, represent one notable type ofmultifunctional nanovector.

Polymer micelles are particularly attractive due to their ability todeliver large payloads of a variety of drugs (e.g. small molecule,proteins, and DNA/RNA therapeutics), their improved in vivo stability ascompared to other colloidal carriers (e.g. liposomes), and theirnanoscopic size which allows for passive accumulation in diseasedtissues, such as solid tumors, by the enhanced permeation and retention(EPR) effect. Using appropriate surface functionality, polymer micellesare further decorated with cell-targeting groups and permeationenhancers that can actively target diseased cells and aid in cellularentry, resulting in improved cell-specific delivery.

While self assembly represents a convenient method for the bottom-updesign of nanovectors, the forces that drive and sustain the assembly ofpolymer micelles are concentration dependent and inherently reversible.In clinical applications, where polymer micelles are rapidly dilutedfollowing administration, this reversibility, along with highconcentrations of micelle-destabilizing blood components (e.g. proteins,lipids, and phospholipids), often leads to premature dissociation of thedrug-loaded micelle before active or passive targeting is effectivelyachieved. For polymer micelles to fully reach their cell-targetingpotential and exploit their envisioned multi-functionality, in vivocirculation time must be improved. Drug delivery vehicles are needed,which are infinitely stable to post-administration dilution, can avoidbiological barriers (e.g. reticuloendothelial system (RES) uptake), anddeliver drugs in response to the physiological environment encounteredin diseased tissues, such as solid tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a representation of a core crosslinked micelle, a shellcrosslinked micelle, and an outer-core crosslinked micelle of thepresent invention.

FIG. 2 depicts an exemplary disulfide crosslinking reaction.

FIG. 3 depicts an exemplary ester crosslinking reaction.

FIG. 4 depicts an exemplary ester crosslinking reaction.

FIG. 5 depicts an exemplary hydrazone crosslinking reaction.

FIG. 6 depicts an exemplary hydrazone crosslinking reaction.

FIG. 7 depicts an exemplary Schiff base crosslinking reaction.

FIG. 8 depicts an exemplary Schiff base crosslinking reaction.

FIG. 9 depicts an exemplary zinc crosslinking reaction.

FIG. 10 depicts an exemplary dual crosslinking reaction.

FIG. 11 shows the CMC experimental data for propyne-aryl-poly(ethyleneglycol)-b-poly(aspartic acid)-b-[poly(phenylalanine)-co-poly(tyrosine)].

FIG. 12 shows a graphical comparison between zinc crosslinked micellesand uncrosslinked control experiments.

FIG. 13 depicts a graph of pyrene loaded crosslinked micelles before andafter the addition of lactic acid.

FIG. 14 shows the conjugation of acetylene-functionalized micelles withazide-containing folate or an azide-containing GRGDS oligopeptide byclick chemistry.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION 1. GeneralDescription:

Bionanotechnology is a broad, multi-disciplinary field encompassing thebiological, chemical, physical, and engineering sciences and isdedicated to the design and manipulation of biomaterials on thenanometer size scale. These “nanodevices” offer the potential to becomehighly advanced, multi-functional tools capable of detection, diagnosis,and personalized treatment of diseases, such as cancer. In the case ofdrug delivery, nanoscopic therapeutic carriers, or “nanovectors,” are apotentially promising method to selectively deliver chemotherapeuticagents to cancerous and other diseased tissue. The advantages ofnano-sized encapsulation devices are numerous. For example, whencompared to single molecule drugs or diagnostic agents, “nanovectors”can transport much larger quantities of such agents. Nanoscopic drugdelivery systems are generally more apt to elude biological barriers,resulting in reduced inactivation or excretion of the encapsulatedtherapeutic. Multi-functionality is a common feature of nanovectorswhereby multiple drugs, diagnostic agents, and targeting groups can bepackaged into a single system. The bottom-up design of nanoscopic drugdelivery systems often involves the precise self assembly of singlemolecules or polymeric units to create complex, multi-functionaldevices.

Polymer micelles are one type of nanovector formed by the aqueousassembly of block copolymers that are polymer chains containing bothhydrophilic and hydrophobic portions. These structures often exist asspherical particles with a core-shell morphology and sub-microndiameter. Their size and structural uniformity impart a strikingresemblance to virus particles, which are Nature's version of theperfect delivery system and are capable of highly efficient delivery tocells and tissue. It is believed that the nanoscopic size of viruses(approximately 20 to 400 nanometers in diameter) contributes to theirability to elude the body's natural defense mechanisms while proteins onthe virus surface enable highly selective targeting and infection ofspecific cells. The design of nanovectors, such as block copolymermicelles, that effectively mimic the selectivity and evasiveness ofviral particles remains a major goal of drug delivery research. Polymermicelles present a viable alternative due to the inherent modularity ofblock copolymers, which offer considerable tuning of the micelle sizeand surface functionality. In certain embodiments, micelles of thepresent invention, as described in detail infra, are about 20 to about200 nanometers in diameter. In other embodiments, micelles of thepresent invention, as described in detail infra, are about 20 to about250 nanometers in diameter.

One advantage of the polymer micelle modularity is the ability to tunethe core and shell components. This is particularly useful for drugdelivery because the core of the assembly can serve as a reservoir for avariety of therapeutic agents while the hydrophilic shell impartssolubility and stability to the aqueous assemblies. From apharmacokinetic viewpoint, the distribution of drug-loaded micelles islargely determined by the size and surface chemistry of the micelle andnot by the drug itself. Thus, polymer micelles possessing a hydrophobiccore are utilized for the encapsulation of potent, small molecule drugsthat were previously shelved due to poor aqueous solubility. Theisolation of hydrophobic chemotherapeutics in the micelle core has alsoprovided new strategies to overcome multi-drug resistance (MDR)mechanisms in cancer cells. Polymer micelles with cationically charged,core-forming blocks are used to encapsulate biomolecules such as plasmidDNA and siRNA. Therapeutics of this type are normally susceptible torapid in vivo degradation, and their encapsulation in polymer micellesimproves their biodistribution profiles thus leading to future clinicalsuccesses.

One biological barrier to any drug delivery system and another issuewhich cell-responsive nanovectors addresess is the non-specific uptakeby the reticuloendothelial system. The RES consists of a host of cellswhich are designed to remove cellular debris and foreign particles fromthe bloodstream. Like viruses, synthetic nanovectors are more apt atescaping RES detection by the nature of their size. In addition, thecovalent attachment of poly(ethylene glycol) is a commonly used methodto reduce opsonization and non-specific RES uptake of small molecule,protein, and nanoparticulate drug carriers. See Harris, J. M.; Martin,N. E.; Modi, M. Clin. Pharmacokin. 2001, 40, 539-551; Bhadra, D.;Bhadra, S.; Jain, P.; Jain, N. K. Pharmazie 2002, 57, 5-29; Shenoy, D.B.; Amiji, M. A. Int. J. Pharm. 2005, 293, 261-270; and Torchilin, V.Adv. Drug Del. Rev. 2002, 54, 235-252.

PEG has become a standard choice for the hydrophilic, corona-formingsegment of block copolymer micelles, and numerous studies have confirmedits ability to reduce RES uptake of micellar delivery systems. See Kwon,G.; Suwa, S.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Kataoka, K. J. Cont.Rel. 1994, 29, 17-23; Caliceti, P.; Veronese, F. M. Adv. Drug Del. Rev.2003, 55, 1261-1277; Ichikawa, K.; Hikita, T.; Maeda, N.; Takeuchi, Y.;Namba, Y.; Oku, N. Bio. Pharm. Bull. 2004, 27, and 443-444. The abilityto tailor PEG chain lengths offers numerous advantages in drug carrierdesign since studies have shown that circulation times and RES uptakeare influenced by the length of the PEG block. In general, longer PEGchains lead to longer circulation times and enhanced stealth properties.In a systematic study of PEG-b-poly(lactic-co-glycolic acid) (PLGA)micelles with PEG molecular weights ranging from 5,000-20,000 Da, Langerand coworkers found that micelles coated with 20,000 Da PEG chains werethe least susceptible to liver uptake. After 5 hours of circulation,less than 30% of the micelles had accumulated in the liver. See Gref,R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.;Langer, R. Science 1994, 263, 1600-1603.

While PEGylation of nanovectors is an effective method to reduce RESuptake and extend in vivo circulation lifetime, other challenges existwhich limit the ultimate effectiveness of colloidal drug carriers. Onesuch barrier relates to their self assembly and subsequent in vivostability. Self assembly represents a convenient, bottom-up approach tonanovector design. The hydrophobic forces that drive the aqueousassembly of colloidal drug carriers, such as polymer micelles andliposomes, are relatively weak, and these assembled structuresdissociate below a finite concentration known as the critical micelleconcentration (“CMC”). The CMC value of these systems is of greatimportance in clinical applications since drug-loaded colloidal carriersare diluted in the bloodstream following administration and rapidlyreach concentrations below the CMC (μM or less) leading to micelledissociation. In addition, non-specific interactions withsurfactant-like components in the blood (e.g. proteins, lipids, etc.)also act to destabilize drug-loaded micelles. See Savić, R.; Azzam, T.;Eisenberg, A.; Maysinger, D. Langmuir 2006, ASAP article. These eventsoften lead to premature drug release outside the targeted area,rendering the drug carrier and cell-targeting strategies ineffective.

Despite the large volume of work on micellar drug carriers, littleeffort has focused on improving their in vivo stability to dilution. Inmost cases, amphiphilic block copolymers lack the functionalitynecessary for post-assembly crosslinking strategies. Wooley andcoworkers have addressed this issue by crosslinking the poly(acrylicacid) corona of the polymer micelles, forming shell-crosslinkednanoparticles. See Thurmond, K. B.; Huang, H. Y.; Clark, C. G.;Kowalewski, T.; Wooley, K. L. Coll. Surf B. 1999, 16, 45-54. Covalentcrosslinking produces nanoparticles with improved stability and offersthe additional benefit of enhanced therapeutic payload since thecore-forming block is chemically removed after crosslinking. See Zhang,Q.; Remsen, E. E.; Wooley, K. L. J. Am. Chem. Soc. 2000, 122, 3642-3651.

In a separate approach, Kataoka and coworkers have developed methods toreversibly crosslink the core of diblock polymer micelles to improvestability. For example, the chemotherapy drug cisplatin was encapsulatedusing PEG-b-poly(aspartic acid) copolymers, forming reversible chemicalbonds in the micelle core. See Nishiyama, N.; Yokoyama, M.; Aoyagi, T.;Okano, T.; Sakurai, Y.; Kataoka, K. Langmuir 1999, 15, 377-383. Themicelles were stable to dilution as determined by dynamic lightscattering studies, and the core-crosslinking was reversible in thepresence of chloride ions, resulting in dissociation of the polymermicelles and release of cisplatin. However, in vivo studies usingtumor-bearing mice showed remarkably fast decay of the cisplatin-loadedmicelles, which resulted in accumulation of the drug in the liver andspleen. See Nishiyama, N.; Kato, Y.; Sugiyama, Y.; Kataoka, K. Pharm.Res. 2001, 18, 1035-1041. Kataoka's group has also reported alternativecore crosslinking strategies that utilize disulfide chemistry. In thiscase, cysteine units were randomly incorporated into the lysine portionof PEG-b-poly(L-lysine) copolymers and used for encapsulation ofantisense RNA. See Kakizawa, Y.; Harada, A.; Kataoka, K. J. Am. Chem.Soc. 1999, 121, 11247-11248; and Kakizawa, Y.; Harada, A.; Kataoka, K.Biomacromolecules 2001, 2, 491-497. The cysteine side chains weresubsequently oxidized in the core to form disulfide crosslinked,RNA-loaded micelles. These micelles were shown to selectively dissociatein the presence of glutathione (GSH), a reducing agent found inappreciable quantities in the cell cytoplasm, offering an effectivemethod for intracellular delivery of the therapeutic. Other corecrosslinking techniques have been devised that utilize polymerend-groups, such as methacrylate and olefmic functionality, which arecrosslinked by free radicals. See Iijima, M.; Nagasaki, Y.; Okada, T.;Kato, M.; Kataoka, K. Macromolecules 1999, 32, 1140-1146; and Tian, L.;Yam, L.; Wang, J. Z.; Tat, H.; Uhrich, K. E. J. Mat. Chem. 2004, 14,2317-2324. One notable disadvantage of the core crosslinking approach isthe inherent reduction of free-volume in the micelle core, whichultimately limits drug loading in the micelle.

Armes and coworkers have used covalent chemistries to crosslink theouter core of micelles made from poly[(ethyleneoxide)-block-2-(dimethylamino)ethylmethacrylate-block-2-(diethylamino)methacrylate]copolymers. The additionof the bifunctional crosslinker, 1,2-bis(2-iodoethoxy)ethane, was shownto effectively crosslink the 2-(dimethylamino)ethyl methacrylate block,forming irreversible quaternary ammonium crosslinks. See Liu, S.;Weaver, J. V. M.; Tang, Y.; Billingham, N. C.; Armes, S. P.Macromolecules, 2002, 35, 6121-6131. McCormick and coworkers havesynthesized poly(ethyleneoxide)-block-[(N,N-dimethylacrylamide)-stat-(N-acryloxysuccinimide)]-block-(N-isopropylacrylamide)copolymerswhere the N-acryloxysuccinimide units are reacted with cystamine tocrosslink the outer core of the micelle through reversible disulfidebonds. See Li, Y.; Lokitz, B. S.; Armes, S. P.; McCormick, C. L.Macromolecules 2006, ASAP article.

To address these pressing issues and develop improved disease-fightingsystems, the present application describes the design and synthesis of“smart,” drug-loaded polymer micelles which are stable to dilution incirculation, can more effectively accumulate in diseased cells, anddissociate in response to the range of environmental changes commonlyfound in diseased tissue and cells.

In certain embodiments, the present invention provides crosslinkedmicelles which effectively encapsulate hydrophobic or ionic therapeuticagents at pH 7.4 (blood) but dissociate and release the drug attargeted, acidic pH values ranging from 5.0 (endosomal pH) to 6.8(extracellular tumor pH). In yet other embodiements, the pH value can beadjusted between 4.0 and 7.4. These pH-targeted nanovectors willdramatically improve the cancer-specific delivery of chemotherapeuticagents and minimize the harmful side effects commonly encountered withpotent chemotherapy drugs. In addition, the utilization of chemistrieswhich can be tailored to dissociate across a range of pH values makethese drug-loaded micelles applicable in treating solid tumors andmalignancies that have become drug resistant.

The pH-responsive block copolymers and polymer micelles described hereinare designed with an emphasis on modularity and multi-functionality.Although the encapsulation and delivery of doxorubicin and camptothecinare exemplified, it is contemplated that the present invention alsoprovides a technology platform whereby a multitude of nanovectors aredesigned and tailored by simple variations in poly(amino acid) type andlength, crosslinking chemistries, and surface targeting functionalities.Examples include polymer micelles with tailored ionic blocks for siRNAand protein encapsulation, reversible metal crosslinking strategieswhich incorporate MRI contrast agents (e.g. iron and gadoliniumderivatives), and the application of micelles with reactive surfacefunctionality for attachment of drugs, permeation enhancers, andtargeting groups.

According to one embodiment, the present invention provides a micellecomprising a multiblock copolymer which comprises a polymerichydrophilic block, a crosslinked poly(amino acid block), and apoly(amino acid block), characterized in that said micelle has an innercore, a crosslinked outer core, and a hydrophilic shell. It will beappreciated that the polymeric hydrophilic block corresponds to thehydrophilic shell, the crosslinked poly(amino acid block) corresponds tothe crosslinked outer core, and the poly(amino acid) block correspondsto the inner core. According to another aspect, the present inventionprovides a drug-loaded micelle comprising a multiblock copolymer whichcomprises a polymeric hydrophilic block, a crosslinked poly(amino acidblock), and a poly(amino acid block), characterized in that said micellehas a drug-loaded inner core, a crosslinked outer core, and ahydrophilic shell.

2. Definitions:

Compounds of this invention include those described generally above, andare further illustrated by the embodiments, sub-embodiments, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

As used herein, the term “sequential polymerization”, and variationsthereof, refers to the method wherein, after a first monomer (e.g. NCA,lactam, or imide) is incorporated into the polymer, thus forming anamino acid “block”, a second monomer (e.g. NCA, lactam, or imide) isadded to the reaction to form a second amino acid block, which processmay be continued in a similar fashion to introduce additional amino acidblocks into the resulting multi-block copolymers.

As used herein, the term “multiblock copolymer” refers to a polymercomprising one synthetic polymer portion and two or more poly(aminoacid) portions. Such multi-block copolymers include those having theformat W-X′-X″, wherein W is a synthetic polymer portion and X and X′are poly(amino acid) chains or “amino acid blocks”. In certainembodiments, the multiblock copolymers of the present invention aretriblock copolymers. As described herein, one or more of the amino acidblocks may be “mixed blocks”, meaning that these blocks can contain amixture of amino acid monomers thereby creating multiblock copolymers ofthe present invention. In some embodiments, the multiblock copolymers ofthe present invention comprise a mixed amino acid block and aretetrablock copolymers.

As used herein, the term “triblock copolymer” refers to a polymercomprising one synthetic polymer portion and two poly(amino acid)portions.

As used herein, the term “tetrablock copolymer” refers to a polymercomprising one synthetic polymer portion and either two poly(amino acid)portions, wherein 1 poly(amino acid) portion is a mixed block or apolymer comprising one synthetic polymer portion and three poly(aminoacid) portions.

As used herein, the term “inner core” as it applies to a micelle of thepresent invention refers to the center of the micelle formed by thesecond (i.e., terminal) poly(amino acid) block. In accordance with thepresent invention, the inner core is not crosslinked. By way ofillustration, in a triblock polymer of the format W-X′-X″, as describedabove, the inner core corresponds to the X″ block. It is contemplatedthat the X″ block can be a mixed block.

As used herein, the term “outer core” as it applies to a micelle of thepresent invention refers to the layer formed by the first poly(aminoacid) block. The outer core lies between the inner core and thehydrophilic shell. In accordance with the present invention, the outercore is either crosslinkable or is cross-linked. By way of illustration,in a triblock polymer of the format W-X′-X″, as described above, theouter core corresponds to the X′ block. It is contemplated that the X′block can be a mixed block.

As used herein, the terms “drug-loaded” and “encapsulated”, andderivatives thereof, are used interchangeably. In accordance with thepresent invention, a “drug-loaded” micelle refers to a micelle having adrug, or therapeutic agent, situated within the core of the micelle.This is also referred to as a drug, or therapeutic agent, being“encapsulated” within the micelle.

As used herein, the term “polymeric hydrophilic block” refers to apolymer that is not a poly(amino acid) and is hydrophilic in nature.Such hydrophilic polymers are well known in the art and includepolyethyleneoxide (also referred to as polyethylene glycol or PEG), andderivatives thereof, poly(N-vinyl-2-pyrolidone), and derivatives therof,poly(N-isopropylacrylamide), and derivatives thereof, poly(hydroxyethylacrylate), and derivatives thereof, poly(hydroxylethyl methacrylate),and derivatives thereof, and polymers ofN-(2-hydroxypropoyl)methacrylamide (HMPA) and derivatives thereof.

As used herein, the term “poly(amino acid)” or “amino acid block” refersto a covalently linked amino acid chain wherein each monomer is an aminoacid unit. Such amino acid units include natural and unnatural aminoacids. In certain embodiments, each amino acid unit is in theL-configuration. Such poly(amino acids) include those having suitablyprotected functional groups. For example, amino acid monomers may havehydroxyl or amino moieties which are optionally protected by a suitablehydroxyl protecting group or a suitable amine protecting group, asappropriate. Such suitable hydroxyl protecting groups and suitable amineprotecting groups are described in more detail herein, infra. As usedherein, an amino acid block comprises one or more monomers or a set oftwo or more monomers. In certain embodiments, an amino acid blockcomprises one or more monomers such that the overall block ishydrophilic. In other embodiments, an amino acid block comprises one ormore monomers such that the overall block is hydrophobic. In still otherembodiments, amino acid blocks of the present invention include randomamino acid blocks, ie blocks comprising a mixture of amino acidresidues.

As used herein, the phrase “natural amino acid side-chain group” refersto the side-chain group of any of the 20 amino acids naturally occuringin proteins. Such natural amino acids include the nonpolar, orhydrophobic amino acids, glycine, alanine, valine, leucine isoleucine,methionine, phenylalanine, tryptophan, and proline. Cysteine issometimes classified as nonpolar or hydrophobic and other times aspolar. Natural amino acids also include polar, or hydrophilic aminoacids, such as tyrosine, serine, threonine, aspartic acid (also known asaspartate, when charged), glutamic acid (also known as glutamate, whencharged), asparagine, and glutamine. Certain polar, or hydrophilic,amino acids have charged side-chains. Such charged amino acids includelysine, arginine, and histidine. One of ordinary skill in the art wouldrecognize that protection of a polar or hydrophilic amino acidside-chain can render that amino acid nonpolar. For example, a suitablyprotected tyrosine hydroxyl group can render that tyroine nonpolar andhydrophobic by virtue of protecting the hydroxyl group.

As used herein, the phrase “unnatural amino acid side-chain group”refers to amino acids not included in the list of 20 amino acidsnaturally occuring in proteins, as described above. Such amino acidsinclude the D-isomer of any of the 20 naturally occuring amino acids.Unnatural amino acids also include homoserine, ornithine, and thyroxine.Other unnatural amino acids side-chains are well know to one of ordinaryskill in the art and include unnatural aliphatic side chains. Otherunnatural amino acids include modified amino acids, including those thatare N-alkylated, cyclized, phosphorylated, acetylated, amidated,azidylated, labelled, and the like.

As used herein, the phrase “living polymer chain-end” refers to theterminus resulting from a polymerization reaction which maintains theability to react further with additional monomer or with apolymerization terminator.

As used herein, the term “termination” refers to attaching a terminalgroup to a polymer chain-end by the reaction of a living polymer with anappropriate compound. Alternatively, the term “termination” may refer toattaching a terminal group to an amine or hydroxyl end, or derivativethereof, of the polymer chain.

As used herein, the term “polymerization terminator” is usedinterchangeably with the term “polymerization terminating agent” andrefers to a compound that reacts with a living polymer chain-end toafford a polymer with a terminal group. Alternatively, the term“polymerization terminator” may refer to a compound that reacts with anamine or hydroxyl end, or derivative thereof, of the polymer chain, toafford a polymer with a terminal group.

As used herein, the term “polymerization initiator” refers to acompound, which reacts with, or whose anion or free base form reactswith, the desired monomer in a manner which results in polymerization ofthat monomer. In certain embodiments, the polymerization initiator isthe compound that reacts with an alkylene oxide to afford a polyalkyleneoxide block. In other embodiments, the polymerization initiator is theamine salt described herein.

The term “aliphatic” or “aliphatic group”, as used herein, denotes ahydrocarbon moiety that may be straight-chain (i.e., unbranched),branched, or cyclic (including fused, bridging, and spiro-fusedpolycyclic) and may be completely saturated or may contain one or moreunits of unsaturation, but which is not aromatic. Unless otherwisespecified, aliphatic groups contain 1-20 carbon atoms. In someembodiments, aliphatic groups contain 1-10 carbon atoms. In otherembodiments, aliphatic groups contain 1-8 carbon atoms. In still otherembodiments, aliphatic groups contain 1-6 carbon atoms, and in yet otherembodiments aliphatic groups contain 1-4 carbon atoms. Suitablealiphatic groups include, but are not limited to, linear or branched,alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as(cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon. This includes any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen, or; a substitutable nitrogen of a heterocyclic ring including═N— as in 3,4-dihydro-2H-pyrrolyl, —NH— as in pyrrolidinyl, or═N(R^(†))— as in N-substituted pyrrolidinyl.

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic,bicyclic, and tricyclic ring systems having a total of five to fourteenring members, wherein at least one ring in the system is aromatic andwherein each ring in the system contains three to seven ring members.The term “aryl” may be used interchangeably with the term “aryl ring”.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted”, whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable”, as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘); —O—(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘); —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘; —(CH) ₂)₄₋₄(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘); —NO₂; —CN;—N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘); —N(R^(∘))C(S)R^(∘);—(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘) ₂;—(CH₂)₀₋₄N(R^(∘))C(OO)OR^(∘)—N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR⁶⁰²₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘); —(CH₂)₀₋₄OC(O)NR^(∘) ₂;—C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘); —(NOR^(∘))R^(∘);—(CH₂)₀₋₄NR^(∘); —(CH₂)₀₋₄S(O)₂R^(∘); —(CH₂)₀₋₄S(O)₂OR^(∘);—(CH₂)₀₋₄OS(O)₂R^(∘); —S(O)₂NR^(∘) ₂; —(CH₂)₀₋₄S(O)R^(∘);—N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘))S(O)₂R^(∘); —N(OR^(∘))R^(∘);—C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘) ₂; —OP(O)(OR^(∘))₂; SiR^(∘) ₃; —(C₁₋₄ straight or branched)alkylene)O—N(R^(∘) ₂; or—(C₁₋₄ straight or branched)alkylene)C(O)O—N(R^(∘) ₂, wherein each R^(∘)may be substituted as defined below and is independently hydrogen, C₁₋₆aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, or, notwithstanding the definitionabove, two independent occurrences of R^(∘), taken together with theirintervening atom(s), form a 3-12-membered saturated, partiallyunsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, which may besubstituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R∘ together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(), -(haloR^()),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(), —(CH₂)₀₋₂CH(OR^())₂; —O(haloR^()), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(),—(CH₂)₀₋₂SR^(), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(),—(CH₂)₀₋₂NR^() ₂, —NO₂, —SiR^() ₃, —OSiR^() ₃, —C(O)SR^(), —(C₁₋₄straight or branched alkylene)C(O)OR^(), or —SSR^() wherein each R^()is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR^()₂, ═NNHC(O)R^(), ═NNHC(O)OR^(), ═NNHS(O)₂R^(), ═NR^(), ═NOR^(),—O(C(R^() ₂))₂₋₃O—, or —S(C(R^() ₂))₂₋₃S—, wherein each independentoccurrence of R^() is selected from hydrogen, C₁₋₆ aliphatic which maybe substituted as defined below, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. Suitabledivalent substituents that are bound to vicinal substitutable carbons ofan “optionally substituted” group include: —O(CR^() ₂)₂₋₃O—, whereineach independent occurrence of R^() is selected from hydrogen, C₁₋₆aliphatic which may be substituted as defined below, or an unsubstituted5-6-membered saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Asuitable tetravalent substituent that is bound to vicinal substitutablemethylene carbons of an “ontionally substituted” group is the dicobalthexacarbonyl cluster represented by

when depicted with the methylenes which bear it.

Suitable substituents on the aliphatic group of R^() include halogen,—R^(), -(haloR^()), —OH, —OR^(), —O(haloR^()), —CN, —C(O)OH,—C(O)OR^(), —NH₂, —NHR^(), —NR^() ₂, or —NO₂, wherein each R^() isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(\), —S(O)₂R^(\), —S(O)₂NR^(\) ₂,—C(S)NR^(\) ₂, —C(NH)NR^(\) ₂, or —N(R^(\))S(O)₂R^(\); wherein eachR^(\) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(\), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* are independentlyhalogen, —R^(\), -(haloR^(\)), —OH, —OR^(\), —O(haloR^(\)), —CN,—C(O)OH, —C(O)OR^(\), —NH₂, —NHR^(\), —NR^(\) ₂, or —NO₂, wherein eachR^(\) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6 membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Protected hydroxyl groups are well known in the art and include thosedescribed in detail in Protecting Groups in Organic Synthesis, T. W.Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999, theentirety of which is incorporated herein by reference. Examples ofsuitably protected hydroxyl groups further include, but are not limitedto, esters, carbonates, sulfonates allyl ethers, ethers, silyl ethers,alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples ofsuitable esters include formates, acetates, proprionates, pentanoates,crotonates, and benzoates. Specific examples of suitable esters includeformate, benzoyl formate, chloroacetate, trifluoroacetate,methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate,pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate,p-benylbenzoate, 2,4,6-trimethylbenzoate. Examples of suitablecarbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl,2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, andp-nitrobenzyl carbonate. Examples of suitable silyl ethers includetrimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triisopropylsilyl ether, and other trialkylsilylethers. Examples of suitable alkyl ethers include methyl, benzyl,p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ether,or derivatives thereof. Alkoxyalkyl ethers include acetals such asmethoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl,benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, andtetrahydropyran-2-yl ether. Examples of suitable arylalkyl ethersinclude benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl,O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl,p-cyanobenzyl, 2- and 4-picolyl ethers.

Protected amines are well known in the art and include those describedin detail in Greene (1999). Suitable mono-protected amines furtherinclude, but are not limited to, aralkylamines, carbamates, allylamines, amides, and the like. Examples of suitable mono-protected aminomoieties include t-butyloxycarbonylamino (—NHBOC),ethyloxycarbonylamino, methyloxycarbonylamino,trichloroethyloxycarbonylamino, allyloxycarbonylamino (-NHAlloc),benzyloxocarbonylamino (-NHCBZ), allylamino, benzyl amino (-NHBn),fluorenylmethylcarbonyl (-NHFmoc), formamido, acetamido,chloroacetamido, dichloroacetamido, trichloroacetamido, phenylacetamido,trifluoroacetamido, benzamido, t-butyldiphenylsilyl, and the like.Suitable di-protected amines include amines that are substituted withtwo substituents independently selected from those described above asmono-protected amines, and further include cyclic imides, such asphthalimide, maleimide, succinimide, and the like. Suitable di-protectedamines also include pyrroles and the like,2,2,5,5-tetramethyl-[1,2,5]azadisilolidine and the like, and azide.

Protected aldehydes are well known in the art and include thosedescribed in detail in Greene (1999). Suitable protected aldehydesfurther include, but are not limited to, acyclic acetals, cyclicacetals, hydrazones, imines, and the like. Examples of such groupsinclude dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzylacetal, bis(2-nitrobenzyl) acetal, 1,3-dioxanes, 1,3-dioxolanes,semicarbazones, and derivatives thereof.

Protected carboxylic acids are well known in the art and include thosedescribed in detail in Greene (1999). Suitable protected carboxylicacids further include, but are not limited to, optionally substitutedC₁₋₆ aliphatic esters, optionally substituted aryl esters, silyl esters,activated esters, amides, hydrazides, and the like. Examples of suchester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,benzyl, and phenyl ester, wherein each group is optionally substituted.Additional suitable protected carboxylic acids include oxazolines andortho esters.

Protected thiols are well known in the art and include those describedin detail in Greene (1999). Suitable protected thiols further include,but are not limited to, disulfides, thioethers, silyl thioethers,thioesters, thiocarbonates, and thiocarbamates, and the like. Examplesof such groups include, but are not limited to, alkyl thioethers, benzyland substituted benzyl thioethers, triphenylmethyl thioethers, andtrichloroethoxycarbonyl thioester, to name but a few.

A “crown ether moiety” is the radical of a crown ether. A crown ether isa monocyclic polyether comprised of repeating units of —CH₂CH₂O—.Examples of crown ethers include 12-crown-4, 15-crown-5, and 18-crown-6.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enrichedcarbon are within the scope of this invention. Such compounds areuseful, for example, as in neutron scattering experiments, as analyticaltools or probes in biological assays.

As used herein, the term “detectable moiety” is used interchangeablywith the term “label” and relates to any moiety capable of beingdetected (e.g., primary labels and secondary labels). A “detectablemoiety” or “label” is the radical of a detectable compound.

“Primary” labels include radioisotope-containing moieties (e.g.,moieties that contain ³²P, ³³P, ³⁵S, or ¹⁴C), mass-tags, and fluorescentlabels, and are signal-generating reporter groups which can be detectedwithout further modifications.

Other primary labels include those useful for positron emissiontomography including molecules containing radioisotopes (e.g. ¹⁸-F) orligands with bound radioactive metals (e.g. ⁶²Cu). In other embodiments,primary labels are contrast agents for magnetic resonance imaging suchas gadolinium, gadolinium chelates, or iron oxide (e.g Fe₃O₄ and Fe₂O₃)particles. Similarly, semiconducting nanoparticles (e.g. cadmiumselenide, cadmium sulfide, cadmium telluride) are useful as fluorescentlabels. Other metal nanoparticles (e.g colloidal gold) also serve asprimary labels.

“Secondary” labels include moieties such as biotin, or protein antigens,that require the presence of a second compound to produce a detectablesignal. For example, in the case of a biotin label, the second compoundmay include streptavidin-enzyme conjugates. In the case of an antigenlabel, the second compound may include an antibody-enzyme conjugate.Additionally, certain fluorescent groups can act as secondary labels bytransferring energy to another compound or group in a process ofnonradiative fluorescent resonance energy transfer (FRET), causing thesecond compound or group to then generate the signal that is detected.

Unless otherwise indicated, radioisotope-containing moieties areoptionally substituted hydrocarbon groups that contain at least oneradioisotope. Unless otherwise indicated, radioisotope-containingmoieties contain from 1-40 carbon atoms and one radioisotope. In certainembodiments, radioisotope-containing moieties contain from 1-20 carbonatoms and one radioisotope.

The terms “fluorescent label”, “fluorescent group”, “fluorescentcompound”, “fluorescent dye”, and “fluorophore”, as used herein, referto compounds or moieties that absorb light energy at a definedexcitation wavelength and emit light energy at a different wavelength.Examples of fluorescent compounds include, but are not limited to: AlexaFluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, AlexaFluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, AlexaFluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL,BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568,BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY650/665), Carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue,Cascade Yellow, Coumarin 343, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5),Dansyl, Dapoxyl, Dialkylaminocoumarin,4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin,Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800),JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin,Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X.

The term “mass-tag” as used herein refers to any moiety that is capableof being uniquely detected by virtue of its mass using mass spectrometry(MS) detection techniques. Examples of mass-tags include electrophorerelease tags such asN-[3-[4′-[(p-Methoxytetrafluorobenzyl)oxy]phenyl]-3-methylglyceronyl]isonipecoticAcid, 4′-[2,3,5,6-Tetrafluoro-4-(pentafluorophenoxyl)]methylacetophenone, and their derivatives. The synthesis and utility of thesemass-tags is described in U.S. Pat. Nos. 4,650,750, 4,709,016,5,360,8191, 5,516,931, 5,602,273, 5,604,104, 5,610,020, and 5,650,270.Other examples of mass-tags include, but are not limited to,nucleotides, dideoxynucleotides, oligonucleotides of varying length andbase composition, oligopeptides, oligosaccharides, and other syntheticpolymers of varying length and monomer composition. A large variety oforganic molecules, both neutral and charged (biomolecules or syntheticcompounds) of an appropriate mass range (100-2000 Daltons) may also beused as mass-tags.

The term “substrate”, as used herein refers to any material ormacromolecular complex to which a functionalized end-group of a blockcopolymer can be attached. Examples of commonly used substrates include,but are not limited to, glass surfaces, silica surfaces, plasticsurfaces, metal surfaces, surfaces containing a metalic or chemicalcoating, membranes (eg., nylon, polysulfone, silica), micro-beads (eg.,latex, polystyrene, or other polymer), porous polymer matrices (eg.,polyacrylamide gel, polysaccharide, polymethacrylate), macromolecularcomplexes (eg., protein, polysaccharide).

3. Description of Exemplary Embodiments

A. Multiblock Copolymers

As described generally above, one embodiment of the present inventionprovides a micelle comprising a multiblock copolymer which comprises apolymeric hydrophilic block, a crosslinked poly(amino acid block), and apoly(amino acid) block, characterized in that said micelle has an innercore, a crosslinked outer core, and a hydrophilic shell.

Amphiphilic multiblock copolymers, as described herein, canself-assemble in aqueous solution to form nano- and micron-sizedstructures. In water, these amphiphilic multiblock copolymers assembleby multi-molecular micellization when present in solution above thecritical micelle concentration (CMC). Without wishing to be bound by anyparticular theory, it is believed that the hydrophobic poly(amino acid)portion or “block” of the copolymer collapses to form the micellar core,while the hydrophilic PEG block forms a peripheral corona and impartswater solubility. In certain embodiments, the multiblock copolymers inaccordance with the present invention possess distinct hydrophobic andhydrophilic segments that form micelles. In addition, these multiblockpolymers comprise a poly(amino acid) block which contains functionalitysuitable for crosslinking. It will be appreciated that thisfunctionality is found on the corresponding amino acid side-chain.

Multiblock copolymers of the present invention contain poly(amino acid)blocks and a water-soluble polymer block. Poly(amino acid) (PAA)segments possess a wide range of functionality and are natural buildingblocks with inherent biocompatibility. In addition, PAA copolymers arehydrolytically stable and can tolerate most chemical transformationconditions yet can be enzymatically degradable.

In certain embodiments, the PEG block possesses a molecular weight ofapprox. 10,000 Da (225 repeat units) and contains at least one terminalamine hydrochloride salt used to initiate the synthesis of poly(aminoacid) multi-block copolymers. Without wishing to be bound by theory, itis believed that this particular PEG chain length imparts adequatewater-solubility to the micelles and provides relatively long in vivocirculation times.

In certain embodiments, the present invention provides a micellecomprising a multiblock copolymer of formula I:

wherein:

n is 10-2500;

m is 1 to 1000;

m′ is 1 to 1000;

R^(x) is a natural or unnatural amino acid side-chain group that iscapable of crosslinking;

R^(y) is a hydrophobic or ionic, natural or unnatural amino acidside-chain group;

R¹ is —Z(CH₂CH₂Y)_(p)(CH₂)_(t)R³, wherein:

-   -   Z is —O—, —S—, or —C≡C—, or —CH₂;    -   each Y is independently —O— or —S—;    -   p is 0-10;    -   t is 0-10; and    -   R³ is —N₃, —CN, a mono-protected amine, a di-protected amine, a        protected aldehyde, a protected hydroxyl, a protected carboxylic        acid, a protected thiol, a 9-30 membered crown ether, or an        optionally substituted group selected from aliphatic, a 5-8        membered saturated, partially unsaturated, or aryl ring having        0-4 heteroatoms independently selected from nitrogen, oxygen, or        sulfur, an 8-10 membered saturated, partially unsaturated, or        aryl bicyclic ring having 0-5 heteroatoms independently selected        from nitrogen, oxygen, or sulfur, or a detectable moiety;    -   Q is a valence bond or a bivalent, saturated or unsaturated,        straight or branched C₁₋₁₂ alkylene chain, wherein 0-6 methylene        units of Q are independently replaced by -Cy-, —O—, —NH—, —S—,        —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO₂—, —SO₂NH—, —NHC(O)—,        —C(O)NH—, —OC(O)NH—, or —NHC(O)O—, wherein: -Cy- is an        optionally substituted 5-8 membered bivalent, saturated,        partially unsaturated, or aryl ring having 0-4 heteroatoms        independently selected from nitrogen, oxygen, or sulfur, or an        optionally substituted 8-10 membered bivalent saturated,        partially unsaturated, or aryl bicyclic ring having 0-5        heteroatoms independently selected from nitrogen, oxygen, or        sulfur;    -   R^(2a) is a mono-protected amine, a di-protected amine, —N(R⁴)₂,        —NR⁴C(O)R⁴, —NR⁴C(O)N(R⁴)₂, —NR⁴C(O)OR⁴, or —NR⁴SO₂R⁴; and    -   each R⁴ is independently an optionally substituted group        selected from hydrogen, aliphatic, a 5-8 membered saturated,        partially unsaturated, or aryl ring having 0-4 heteroatoms        independently selected from nitrogen, oxygen, or sulfur, an 8-10        membered saturated, partially unsaturated, or aryl bicyclic ring        having 0-5 heteroatoms independently selected from nitrogen,        oxygen, or sulfur, or a detectable moiety, or:        -   two R⁴ on the same nitrogen atom are taken together with            said nitrogen atom to form an optionally substituted 4-7            membered saturated, partially unsaturated, or aryl ring            having 1-4 heteroatoms independently selected from nitrogen,            oxygen, or sulfur.

According to another embodiment, the present invention providescompounds of formula I, as described above, wherein said compounds havea polydispersity index (“PDI”) of about 1.0 to about 1.2. According toanother embodiment, the present invention provides compounds of formulaI, as described above, wherein said compound has a polydispersity index(“PDI”) of about 1.03 to about 1.15. According to yet anotherembodiment, the present invention provides compounds of formula I, asdescribed above, wherein said compound has a polydispersity index(“PDI”) of about 1.10 to about 1.20. According to other embodiments, thepresent invention provides compounds of formula I having a PDI of lessthan about 1.10.

In certain embodiments, the present invention provides compounds offormula I, as described above, wherein n is about 225. In otherembodiments, n is about 200 to about 300. In still other embodiments, nis about 200 to about 250. In still other embodiments, n is about 100 toabout 150. In still other embodiments, n is about 400 to about 500. Inother embodiments, n is about 10 to about 40. In other embodiments, n isabout 40 to about 60. In still other embodiments, n is about 90 to about150. In still other embodiments, n is about 200 to about 250. In otherembodiments, n is about 300 to about 375. In still other embodiments, nis about 650 to about 750.

In certain embodiments, the m′ group of formula I is about 5 to about500. In certain embodiments, the m′ group of formula I is about 10 toabout 250. In other embodiments, m′ is about 10 to about 50. Accordingto yet another embodiment, m′ is about 15 to about 40. In otherembodiments, m′ is about 20 to about 40. According to yet anotherembodiment, m′ is about 50 to about 75. According to other embodiments,m and m′ are independently about 10 to about 100. In certainembodiments, m is 5-50. In other embodiments, m is 5-25. In certainembodiments, m′ is 5-50. In other embodiments, m′ is 5-10. In otherembodiments, m′ is 10-20. In certain embodiments, m and m′ add up toabout 30 to about 60. In still other embodiments, m is 1-20 repeat unitsand m′ is 10-50 repeat units.

In certain embodiments, the R³ moiety of the R¹ group of formula I is—N₃.

In other embodiments, the R³ moiety of the R¹ group of formula I is —CN.

In still other embodiments, the R³ moiety of the R¹ group of formula Iis a mono-protected amine or a di-protected amine.

In certain embodiments, the R³ moiety of the R¹ group of formula I is anoptionally substituted aliphatic group. Examples include t-butyl,5-norbornene-2-yl, octane-5-yl, acetylenyl, trimethylsilylacetylenyl,triisopropylsilylacetylenyl, and t-butyldimethylsilylacetylenyl. In someembodiments, said R³ moiety is an optionally substituted alkyl group. Inother embodiments, said R³ moiety is an optionally substituted alkynylor alkenyl group. When said R³ moiety is a substituted aliphatic group,suitable substituents on R³ include CN, N₃, trimethylsilyl,triisopropylsilyl, t-butyldimethylsilyl, N-methyl propiolamido,N-methyl-4-acetylenylanilino, N-methyl-4-acetylenylbenzoamido,bis-(4-ethynyl-benzyl)-amino, dipropargylamino, di-hex-5-ynyl-amino,di-pent-4-ynyl-amino, di-but-3-ynyl-amino, propargyloxy, hex-5-ynyloxy,pent-4-ynyloxy, di-but-3-ynyloxy, N-methyl-propargylamino,N-methyl-hex-5-ynyl-amino, N-methyl-pent-4-ynyl-amino,N-methyl-but-3-ynyl-amino, 2-hex-5-ynyldisulfanyl,2-pent-4-ynyldisulfanyl, 2-but-3-ynyldisulfanyl, and2-propargyldisulfanyl. In certain embodiments, the R¹ group is2-(N-methyl-N-(ethynylcarbonyl)amino)ethoxy, 4-ethynylbenzyloxy, or2-(4-ethynylphenoxy)ethoxy.

In certain embodiments, the R³ moiety of the R¹ group of formula I is anoptionally substituted aryl group. Examples include optionallysubstituted phenyl and optionally substituted pyridyl. When said R³moiety is a substituted aryl group, suitable substituents on R³ includeCN, N₃, NO₂, —CH₃, —CH₂N₃, —CH═CH₂, —C≡CH, Br, I, F,bis-(4-ethynyl-benzyl)-amino, dipropargylamino, di-hex-5-ynyl-amino,di-pent-4-ynyl-amino, di-but-3-ynyl-amino, propargyloxy, hex-5-ynyloxy,pent-4-ynyloxy, di-but-3-ynyloxy, 2-hex-5-ynyloxy-ethyldisulfanyl,2-pent-4-ynyloxy-ethyldisulfanyl, 2-but-3-ynyloxy-ethyldisulfanyl,2-propargyloxy-ethyldisulfanyl, bis-benzyloxy-methyl,[1,3]dioxolan-2-yl, and [1,3]dioxan-2-yl.

In other embofiments, the R³ moiety is an aryl group substituted with asuitably protected amino group. According to another aspect, the R³moiety is phenyl substituted with a suitably protected amino group.

In other embodiments, the R³ moiety of the R¹ group of formula I is aprotected hydroxyl group. In certain embodiments the protected hydroxylof the R³ moiety is an ester, carbonate, sulfonate, allyl ether, ether,silyl ether, alkyl ether, arylalkyl ether, or alkoxyalkyl ether. Incertain embodiments, the ester is a formate, acetate, proprionate,pentanoate, crotonate, or benzoate. Exemplary esters include formate,benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate,4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate(trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate,p-benylbenzoate, 2,4,6-trimethylbenzoate. Exemplary carbonates include9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl,2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonate.Examples of suitable silyl ethers include trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl ether, andother trialkylsilyl ethers. Exemplary alkyl ethers include methyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allylether, or derivatives thereof. Exemplary alkoxyalkyl ethers includeacetals such as methoxymethyl, methylthiomethyl,(2-methoxyethoxy)methyl, benzyloxymethyl,beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyran-2-yl ether.Exemplary arylalkyl ethers include benzyl, p-methoxybenzyl (MPM),3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl ethers.

In certain embodiments, the R³ moiety of the R¹ group of formula I is amono-protected or di-protected amino group. In certain embodiments R³ isa mono-protected amine. In certain embodiments R³ is a mono-protectedamine selected from aralkylamines, carbamates, allyl amines, or amides.Exemplary mono-protected amino moieties include t-butyloxycarbonylamino,ethyloxycarbonylamino, methyloxycarbonylamino,trichloroethyloxy-carbonylamino, allyloxycarbonylamino,benzyloxocarbonylamino, allylamino, benzylamino,fluorenylmethylcarbonyl, formamido, acetamido, chloroacetamido,dichloroacetamido, trichloroacetamido, phenylacetamido,trifluoroacetamido, benzamido, and t-butyldiphenylsilylamino. In otherembodiments R³ is a di-protected amine. Exemplary di-protected aminesinclude di-benzylamine, di-allylamine, phthalimide, maleimide,succinimide, pyrrole, 2,2,5,5-tetramethyl-[1,2,5]azadisilolidine, andazide. In certain embodiments, the R³ moiety is phthalimido. In otherembodiments, the R³ moiety is mono- or di-benzylamino or mono- ordi-allylamino. In certain embodiments, the R¹ group is2-dibenzylaminoethoxy.

In other embodiments, the R³ moiety of the R¹ group of formula I is aprotected aldehyde group. In certain embodiments the protected aldehydomoiety of R³ is an acyclic acetal, a cyclic acetal, a hydrazone, or animine. Exemplary R³ groups include dimethyl acetal, diethyl acetal,diisopropyl acetal, dibenzyl acetal, bis(2-nitrobenzyl) acetal,1,3-dioxane, 1,3-dioxolane, and semicarbazone. In certain embodiments,R³ is an acyclic acetal or a cyclic acetal. In other embodiments, R³ isa dibenzyl acetal.

In yet other embodiments, the R³ moiety of the R¹ group of formula I isa protected carboxylic acid group. In certain embodiments, the protectedcarboxylic acid moiety of R³ is an optionally substituted ester selectedfrom C₁₋₆ aliphatic or aryl, or a silyl ester, an activated ester, anamide, or a hydrazide. Examples of such ester groups include methyl,ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, and phenyl ester. Inother embodiments, the protected carboxylic acid moiety of R³ is anoxazoline or an ortho ester. Examples of such protected carboxylic acidmoieties include oxazolin-2-yl and 2-methoxy-[1,3]dioxin-2-yl. Incertain embodiments, the R¹ group is oxazolin-2-ylmethoxy or2-oxazolin-2-yl-1-propoxy.

According to another embodiments, the R³ moiety of the R¹ group offormula I is a protected thiol group. In certain embodiments, theprotected thiol of R³ is a disulfide, thioether, silyl thioether,thioester, thiocarbonate, or a thiocarbamate. Examples of such protectedthiols include triisopropylsilyl thioether, t-butyldimethylsilylthioether, t-butyl thioether, benzyl thioether, p-methylbenzylthioether, triphenylmethyl thioether, and p-methoxyphenyldiphenylmethylthioether. In other embodiments, R³ is an optionally substitutedthioether selected from alkyl, benzyl, or triphenylmethyl, ortrichloroethoxycarbonyl thioester. In certain embodiments, R³ is—S—S-pyridin-2-yl, —S—SBn, —S—SCH₃, or —S—S(p-ethynylbenzyl). In otherembodmients, R³ is —S—S-pyridin-2-yl. In still other embodiments, the R¹group is 2-triphenylmethylsulfanyl-ethoxy.

In certain embodiments, the R³ moiety of the R¹ group of formula I is acrown ether. Examples of such crown ethers include 12-crown-4,15-crown-5, and 18-crown-6.

In still other embodiments, the R³ moiety of the R¹ group of formula Iis a detectable moiety. According to one aspect of the invention, the R³moiety of the R¹ group of formula I is a fluorescent moiety. Suchfluorescent moieties are well known in the art and include coumarins,quinolones, benzoisoquinolones, hostasol, and Rhodamine dyes, to namebut a few. Exemplary fluorescent moieties of the R³ group of R¹ includeanthracen-9-yl, pyren-4-yl, 9-H-carbazol-9-yl, the carboxylate ofrhodamine B, and the carboxylate of coumarin 343. In certainembodiments, the R³ moiety of the R¹ group of formula I is a detectablemoiety selected from:

In certain embodiments, the R³ moiety of the R¹ group of formula I is agroup suitable for Click chemistry. Click reactions tend to involvehigh-energy (“spring-loaded”) reagents with well-defined reactioncoordinates, giving rise to selective bond-forming events of wide scope.Examples include the nucleophilic trapping of strained-ringelectrophiles (epoxide, aziridines, aziridinium ions, episulfoniumions), certain forms of carbonyl reactivity (aldehydes and hydrazines orhydroxylamines, for example), and several types of cycloadditionreactions. The azide-alkyne 1,3-dipolar cycloaddition is one suchreaction. Click chemistry is known in the art and one of ordinary skillin the art would recognize that certain R³ moieties of the presentinvention are suitable for Click chemistry.

Compounds of formula I having R³ moieties suitable for Click chemistryare useful for conjugating said compounds to biological systems ormacromolecules such as proteins, viruses, and cells, to name but a few.The Click reaction is known to proceed quickly and selectively underphysiological conditions. In contrast, most conjugation reactions arecarried out using the primary amine functionality on proteins (e.g.lysine or protein end-group). Because most proteins contain a multitudeof lysines and arginines, such conjugation occurs uncontrollably atmultiple sites on the protein. This is particularly problematic whenlysines or arginines are located around the active site of an enzyme orother biomolecule. Thus, another embodiment of the present inventionprovides a method of conjugating the R¹ groups of a compound of formulaI to a macromolecule via Click chemistry. Yet another embodiment of thepresent invention provides a macromolecule conjugated to a compound offormula I via the R¹ group.

According to one embodiment, the R³ moiety of the R¹ group of formula Iis an azide-containing group. According to another embodiment, the R³moiety of the R¹ group of formula I is an alkyne-containing group. Incertain embodiments, the R³ moiety of the R¹ group of formula I has aterminal alkyne moiety. In other embodiments, R³ moiety of the R¹ groupof formula I is an alkyne moiety having an electron withdrawing group.Accordingly, in such embodiments, the R³ moiety of the R¹ group offormula I is

wherein E is an electron withdrawing group and y is 0-6. Such electronwithdrawing groups are known to one of ordinary skill in the art. Incertain embodiments, E is an ester. In other embodiments, the R³ moietyof the R¹ group of formula I is

wherein E is an electron withdrawing group, such as a —C(O)O— group andy is 0-6.

As defined generally above, Q is a valence bond or a bivalent, saturatedor unsaturated, straight or branched C₁₋₁₂ alkylene chain, wherein 0-6methylene units of Q are independently replaced by -Cy-, —O—, —NH—, —S—,—OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO₂—, —NHSO₂—, —SO₂NH—, —NHC(O)—,—C(O)NH—, —OC(O)NH—, or —NHC(O)O—, wherein -Cy- is an optionallysubstituted 5-8 membered bivalent, saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or an optionally substituted 8-10 membered bivalentsaturated, partially unsaturated, or aryl bicyclic ring having 0-5heteroatoms independently selected from nitrogen, oxygen, or sulfur. Incertain embodiments, Q is a valence bond. In other embodiments, Q is abivalent, saturated C₁₋₁₂ alkylene chain, wherein 0-6 methylene units ofQ are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—,or —C(O)—, wherein -Cy- is an optionally substituted 5-8 memberedbivalent, saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, oran optionally substituted 8-10 membered bivalent saturated, partiallyunsaturated, or aryl bicyclic ring having 0-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, Q is -Cy- (i.e. a C₁ alkylene chain wherein themethylene unit is replaced by -Cy-), wherein -Cy- is an optionallysubstituted 5-8 membered bivalent, saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. According to one aspect of the present invention,-Cy- is an optionally substituted bivalent aryl group. According toanother aspect of the present invention, -Cy- is an optionallysubstituted bivalent phenyl group. In other embodiments, -Cy- is anoptionally substituted 5-8 membered bivalent, saturated carbocyclicring. In still other embodiments, -Cy- is an optionally substituted 5-8membered bivalent, saturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. Exemplary -Cy-groups include bivalent rings selected from phenyl, pyridyl,pyrimidinyl, cyclohexyl, cyclopentyl, or cyclopropyl.

In certain embodiments, R^(x) is a crosslinkable amino acid side-chaingroup and R^(y) is a hydrophobic amino acid side-chain group. Suchcrosslinkable amino acid side-chain groups include tyrosine, serine,cysteine, threonine, aspartic acid (also known as aspartate, whencharged), glutamic acid (also known as glutamate, when charged),asparagine, histidine, lysine, arginine, and glutamine. Such hydrophobicamino acid side-chain groups include a suitably protected tyrosineside-chain, a suitably protected serine side-chain, a suitably protectedthreonine side-chain, phenylalanine, alanine, valine, leucine,tryptophan, proline, benzyl and alkyl glutamates, or benzyl and alkylaspartates or mixtures thereof. In other embodiments, R^(y) is an ionicamino acid side-chain group. Such ionic amino acid side chain groupsincludes a lysine side-chain, arginine side-chain, or a suitablyprotected lysine or arginine side-chain, an aspartic acid side chain,glutamic acid side-chain, or a suitably protected aspartic acid orglutamic acid side-chain. One of ordinary skill in the art wouldrecognize that protection of a polar or hydrophilic amino acidside-chain can render that amino acid nonpolar. For example, a suitablyprotected tyrosine hydroxyl group can render that tyrosine nonpolar andhydrophobic by virtue of protecting the hydroxyl group. Suitableprotecting groups for the hydroxyl, amino, and thiol, and carboylatefunctional groups of R^(x) and R^(y) are as described herein.

In other embodiments, R^(y) comprises a mixture of hydrophobic andhydrophilic amino acid side-chain groups such that the overallpoly(amino acid) block comprising R^(y) is hydrophobic. Such mixtures ofamino acid side-chain groups include phenylalanine/tyrosine,phenalanine/serine, leucine/tyrosine, and the like. According to anotherembodiment, R^(y) is a hydrophobic amino acid side-chain group selectedfrom phenylalanine, alanine, or leucine, and one or more of tyrosine,serine, or threonine.

As defined above, R^(x) is a natural or unnatural amino acid side-chaingroup capable of forming cross-links. It will be appreciated that avariety of amino acid side-chain functional groups are capable of suchcross-linking, including, but not limited to, carboxylate, hydroxyl,thiol, and amino groups. Examples of IV moieties having functionalgroups capable of forming cross-links include a glutamic acidside-chain, —CH₂C(O)CH, an aspartic acid side-chain, —CH₂CH₂C(O)OH, acystein side-chain, —CH₂SH, a serine side-chain, —CH₂OH, an aldehydecontaining side-chain, —CH₂C(O)H, a lysine side-chain, —(CH₂)₄NH₂, anarginine side-chain, —(CH₂)₃NHC(═NH)NH₂, a histidine side-chain,—CH₂-imidazol-4-yl.

As defined generally above, the R^(2a) group of formula I is amono-protected amine, a di-protected amine, —NHR⁴, —N(R⁴)₂, —NHC(O)R⁴,—NR⁴C(O)R⁴, —NHC(O)NHR⁴, —NHC(O)N(R⁴)₂, —NR⁴C(O)NHR⁴, —NR⁴C(O)N(R⁴)₂,—NHC(O)OR⁴, —NR⁴C(O)OR⁴, —NHSO₂R⁴, or —NR⁴SO₂R⁴, wherein each R⁴ isindependently an optionally substituted group selected from aliphatic, a5-8 membered saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, an8-10-membered saturated, partially unsaturated, or aryl bicyclic ringhaving 0-5 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or a detectable moiety, or two R⁴ on the same nitrogen atom aretaken together with said nitrogen atom to form an optionally substituted4-7 membered saturated, partially unsaturated, or aryl ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, the R^(2a) group of formula I is —NHR⁴ or—N(R⁴)₂ wherein each R⁴ is an optionally substituted aliphatic group.One exemplary R⁴ group is 5-norbornen-2-yl-methyl. According to yetanother aspect of the present invention, the R^(2a) group of formula Iis —NHR⁴ wherein R⁴ is a C₁₋₆ aliphatic group substituted with N₃.Examples include —CH₂N₃. In some embodiments, R⁴ is an optionallysubstituted C₁₋₆ alkyl group. Examples include methyl, ethyl, propyl,butyl, pentyl, hexyl, 2-(tetrahydropyran-2-yloxy)ethyl,pyridin-2-yldisulfanylmethyl, methyldisulfanylmethyl,(4-acetylenylphenyl)methyl, 3-(methoxycarbonyl)-prop-2-ynyl,methoxycarbonylmethyl,2-(N-methyl-N-(4-acetylenylphenyl)carbonylamino)-ethyl,2-phthalimidoethyl, 4-bromobenzyl, 4-chlorobenzyl, 4-fluorobenzyl,4-iodobenzyl, 4-propargyloxybenzyl, 2-nitrobenzyl,4-(bis-4-acetylenylbenzyl)aminomethyl-benzyl, 4-propargyloxy-benzyl,4-dipropargylamino-benzyl, 4-(2-propargyloxy-ethyldisulfanyl)benzyl,2-propargyloxy-ethyl, 2-propargyldisulfanyl-ethyl, 4-propargyloxy-butyl,2-(N-methyl-N-propargylamino)ethyl, and2-(2-dipropargylaminoethoxy)-ethyl. In other embodiments, R⁴ is anoptionally substituted C₂₋₆ alkenyl group. Examples include vinyl,allyl, crotyl, 2-propenyl, and but-3-enyl. When R⁴ group is asubstituted aliphatic group, suitable substituents on R⁴ include N₃, CN,and halogen. In certain embodiments, R⁴ is —CH₂CN, —CH₂CH₂CN,—CH₂CH(OCH₃)₂, 4-(bisbenzyloxymethyl)phenylmethyl, and the like.

According to another aspect of the present invention, the R^(2a) groupof formula I is —NHR⁴ wherein R⁴ is an optionally substituted C₂₋₆alkynyl group. Examples include —CC≡CH, —CH₂C≡CH, —CH₂C≡CCH₃, and—CH₂CH₂C≡CH.

In certain embodiments, the R^(2a) group of formula I is —NHR⁴ whereinR⁴ is an optionally substituted 5-8-membered aryl ring. In certainembodiments, R⁴ is optionally substituted phenyl or optionallysubstituted pyridyl. Examples include phenyl,4-t-butoxycarbonylaminophenyl, 4-azidomethylphenyl,4-propargyloxyphenyl, 2-pyridyl, 3-pyridyl, and 4-pyridyl. In certainembodiments, R^(2a) is 4-t-butoxycarbonylaminophenylamino,4-azidomethylphenamino, or 4-propargyloxyphenylamino.

In certain embodiments, the R^(2a) group of formula I is —NHR⁴ whereinR⁴ is an optionally substituted phenyl ring. Suitable substituents onthe R⁴ phenyl ring include halogen; —(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘); —NO₂; —CN;—N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₀₋₄N(R^(∘))C(O)R^(∘); —N(R^(∘))C(S)R^(∘);—(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘)C(S)NR) ^(∘) ₂;—(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)O R^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₀₋₄C(O)OSiR^(∘) ₃_l ; —(CH₂)₀₋₄OC(O)R^(∘);—(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘) ₂; —C(S)NR^(∘) ₂;—(CH₂)₀₋₄OC(O)NR^(∘) ₂; —C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘);—C(O)CH₂C(O)R^(∘); —C(NOR^(∘)R) ^(∘); —(CH₂)₀₋₄SSR^(∘);—(CH₂)₀₋₄S(O)₂R^(∘); —(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘);—S(O)₂NR^(∘) ₂; —(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂;—N(R^(∘))S(O)₂R^(∘); —N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘) ₂; SiR^(∘) ₃; wherein each independentoccurrence of R^(∘) is as defined herein supra. In other embodiments,the R^(2a) group of formula I is —NHR⁴ wherein R⁴ is phenyl substitutedwith one or more optionally substituted C₁₋₆ aliphatic groups. In stillother embodiments, R⁴ is phenyl substituted with vinyl, allyl,acetylenyl, —CH₂N₃, —CH₂CH₂N₃, —CH₂C≡CCH₃, or —CH₂C≡CH.

In certain embodiments, the R^(2a) group of formula I is -NHR⁴ whereinR⁴ is phenyl substituted with N₃, N(R^(∘) ₂, CO₂R^(∘), orC(O)R^(∘)wherein each R° is independently as defin herein supra.

In certain embodiments, the R^(2a) group of formula I is —N(R⁴)₂ whereineach R⁴ is independently an optionally substituted group selected fromaliphatic, phenyl, naphthyl, a 5-6 membered aryl ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, ora 8-10 membered bicyclic aryl ring having 1-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, or a detectable moiety.

In other embodiments, the R^(2a) group of formula I is —N(R⁴)₂ whereinthe two R⁴ groups are taken together with said nitrogen atom to form anoptionally substituted 4-7 membered saturated, partially unsaturated, oraryl ring having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. According to another embodiment, the two R⁴ groupsare taken together to form a 5-6-membered saturated or partiallyunsaturated ring having one nitrogen wherein said ring is substitutedwith one or two oxo groups. Such R^(2a) groups include, but are notlimited to, phthalimide, maleimide and succinimide.

In certain embodiments, the R^(2a) group of formula I is amono-protected or di-protected amino group. In certain embodimentsR^(2a) is a mono-protected amine. In certain embodiments R^(2a) is amono-protected amine selected from aralkylamines, carbamates, allylamines, or amides. Exemplary mono-protected amino moieties includet-butyloxycarbonylamino, ethyloxycarbonylamino, methyloxycarbonylamino,trichloroethyloxy-carbonylamino, allyloxycarbonylamino,benzyloxocarbonylamino, allylamino, benzylamino,fluorenylmethylcarbonyl, formamido, acetamido, chloroacetamido,dichloroacetamido, trichloroacetamido, phenylacetamido,trifluoroacetamido, benzamido, and t-butyldiphenylsilylamino. In otherembodiments R^(2a) is a di-protected amine. Exemplary di-protected aminomoieties include di-benzylamino, di-allylamino, phthalimide, maleimido,succinimido, pyrrolo, 2,2,5,5-tetramethyl-[1,2,5]azadisilolidino, andazido. In certain embodiments, the R^(2a) moiety is phthalimido. Inother embodiments, the R^(2a) moiety is mono- or di-benzylamino or mono-or di-allylamino.

In certain embodiments, the R^(2a) group of formula I comprises a groupsuitable for Click chemistry. One of ordinary skill in the art wouldrecognize that certain R^(2a) groups of the present invention aresuitable for Click chemistry.

Compounds of formula I having R^(2a) groups comprising groups suitablefor Click chemistry are useful for conjugating said compounds tobiological systems such as proteins, viruses, and cells, to name but afew. After conjugation to a biomolecule, drug, cell, substrate, or thelike, the other end-group functionality, corresponding to the R¹ moietyof formula I, can be used to attach targeting groups for cell specificdelivery including, but not limited to, fluorescent dyes, covalentattachment to surfaces, and incorporation into hydrogels. Thus, anotherembodiment of the present invention provides a method of conjugating theR^(2a) group of a compound of formula I to a fluorescent dye, smallmolecule drug, or macromolecule via Click chemistry. Yet anotherembodiment of the present, invention provides a macromolecule conjugatedto a compound of formula I via the R^(2a) group.

According to one embodiment, the R^(2a) group of formula I is anazide-containing group. According to another embodiment, the R^(2a)group of formula I is an alkyne-containing group.

In certain embodiments, the R^(2a) group of formula I has a terminalalkyne moiety. In other embodiments, the R^(2a) group of formula I is analkyne-containing moiety having an electron withdrawing group.Accordingly, in such embodiments, the R^(2a) group of formula I is

wherein E is an electron withdrawing group and y is 0-6. Such electronwithdrawing groups are known to one of ordinary skill in the art. Incertain embodiments, E is an ester. In other embodiments, the R^(2a)group of formula I is

wherein E is an electron withdrawing group, such as a —C(O)O— group andy is 0-6.

In other embodiments, the present invention provides a micellecomprising a multiblock copolymer of formula II:

wherein:

n is 10-2500;

m is 1 to 1000;

m′ is 1 to 1000;

R^(x) is a crosslinked natural or unnatural amino acid side-chain group;

R^(y) is a hydrophobic or ionic, natural or unnatural, amino acidside-chain group; R¹ is —Z(CH₂CH₂Y)_(p)(CH₂)_(t)R³, wherein:

-   -   -   Z is —O—, —S—, —C≡C, or —CH₂—;        -   each Y is independently —O— or —S—;        -   p is 0-10;        -   t is 0-10; and        -   R³ is —N₃, —CN, a mono-protected amine, a di-protected            amine, a protected aldehyde, a protected hydroxyl, a            protected carboxylic acid, a protected thiol, a 9-30            membered crown ether, or an optionally substituted group            selected from aliphatic, a 5-8 membered saturated, partially            unsaturated, or aryl ring having 0-4 heteroatoms            independently selected from nitrogen, oxygen, or sulfur, an            8-10 membered saturated, partially unsaturated, or aryl            bicyclic ring having 0-5 heteroatoms independently selected            from nitrogen, oxygen, or sulfur, or a detectable moiety;        -   Q is a valence bond or a bivalent, saturated or unsaturated,            straight or branched C₁₋₁₂ alkylene chain, wherein 0-6            methylene units of Q are independently replaced by -Cy-,            —O—, —NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO₂—,            —NHSO₂—, —SO₂NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or            —NHC(O)O—, wherein:            -   -Cy- is an optionally substituted 5-8 membered bivalent,                saturated, partially unsaturated, or aryl ring having                0-4 heteroatoms independently selected from nitrogen,                oxygen, or sulfur, or an optionally substituted 8-10                membered bivalent saturated, partially unsaturated, or                aryl bicyclic ring having 0-5 heteroatoms independently                selected from nitrogen, oxygen, or sulfur;

    -   R^(2a) is a mono-protected amine, a di-protected amine, —N(R⁴)₂,        —NR⁴C(O)R⁴, —NR⁴C(O)N(R⁴)₂, —NR⁴C(O)OR⁴, or —NR⁴SO₂R⁴; and

    -   each R⁴ is independently an optionally substituted group        selected from hydrogen, aliphatic, a 5-8 membered saturated,        partially unsaturated, or aryl ring having 0-4 heteroatoms        independently selected from nitrogen, oxygen, or sulfur, an 8-10        membered saturated, partially unsaturated, or aryl bicyclic ring        having 0-5 heteroatoms independently selected from nitrogen,        oxygen, or sulfur, or a detectable moiety, or:        -   two R⁴ on the same nitrogen atom are taken together with            said nitrogen atom to form an optionally substituted 4-7            membered saturated, partially unsaturated, or aryl ring            having 1-4 heteroatoms independently selected from nitrogen,            oxygen, or sulfur.

According to another embodiment, the present invention providescompounds of formula II, as described above, wherein said compounds havea polydispersity index (“PDI”) of about 1.0 to about 1.2. According toanother embodiment, the present invention provides compounds of formulaII, as described above, wherein said compound has a polydispersity index(“PDI”) of about 1.03 to about 1.15. According to yet anotherembodiment, the present invention provides compounds of formula II, asdescribed above, wherein said compound has a polydispersity index(“PDI”) of about 1.10 to about 1.20. According to other embodiments, thepresent invention provides compounds of formula II having a PDI of lessthan about 1.10.

As defined generally above, the n group of formula II is 10-2500. Incertain embodiments, the present invention provides compounds of formulaII, as described above, wherein n is about 225. In other embodiments, nis about 10 to about 40. In other embodiments, n is about 40 to about60. In still other embodiments, n is about 90 to about 150. In stillother embodiments, n is about 200 to about 250. In other embodiments, nis about 300 to about 375. In other embodiments, n is about 400 to about500. In still other embodiments, n is about 650 to about 750.

In certain embodiments, the m′ group of formula II is about 5 to about500. In certain embodiments, the m′ group of formula II is about 10 toabout 250. In other embodiments, m′ is about 10 to about 50. In otherembodiments, m′ is about 20 to about 40. According to yet anotherembodiment, m′ is about 50 to about 75. According to other embodiments,m and m′ are independently about 10 to about 100. In certainembodiments, m′ is 5-50. In other embodiments, m′ is 5-10. In otherembodiments, m′ is 10-20. In certain embodiments, m and m′ add up toabout 30 to about 60. In still other embodiments, m is 1-20 repeat unitsand m′ is 10-50 repeat units.

In certain embodiments, the R³ moiety of the R¹ group of formula II is—N₃.

In other embodiments, the R³ moiety of the R¹ group of formula II is—CN.

In still other embodiments, the R³ moiety of the R¹ group of formula IIis a mono-protected amine or a di-protected amine.

In certain embodiments, the R³ moiety of the R¹ group of formula II isan optionally substituted aliphatic group. Examples include t-butyl,5-norbornene-2-yl, octane-5-yl, acetylenyl, trimethylsilylacetylenyl,triisopropylsilylacetylenyl, and t-butyldimethylsilylacetylenyl. In someembodiments, said R³ moiety is an optionally substituted alkyl group. Inother embodiments, said R³ moiety is an optionally substituted alkynylor alkenyl group. When said R³ moiety is a substituted aliphatic group,suitable substituents on R³ include CN, N₃, trimethylsilyl,triisopropylsilyl, t-butyldimethylsilyl, N-methyl propiolamido,N-methyl-4-acetylenylanilino, N-methyl-4-acetylenylbenzoamido,bis-(4-ethynyl-benzyl)-amino, dipropargylamino, di-hex-5-ynyl-amino,di-pent-4-ynyl-amino, di-but-3-ynyl-amino, propargyloxy, hex-5-ynyloxy,pent-4-ynyloxy, di-but-3-ynyloxy, N-methyl-propargylamino,N-methyl-hex-5-ynyl-amino, N-methyl-pent-4-ynyl-amino,N-methyl-but-3-ynyl-amino, 2-hex-5-ynyldisulfanyl,2-pent-4-ynyldisulfanyl, 2-but-3-ynyldisulfanyl, and2-propargyldisulfanyl. In certain embodiments, the R¹ group is2-(N-methyl-N-(ethynylcarbonyl)amino)ethoxy, 4-ethynylbenzyloxy, or2-(4-ethynylphenoxy)ethoxy.

In certain embodiments, the R³ moiety of the R¹ group of formula II isan optionally substituted aryl group. Examples include optionallysubstituted phenyl and optionally substituted pyridyl. When said R³moiety is a substituted aryl group, suitable substituents on R³ includeCN, N₃, NO₂, —CH₃, —CH₂N₃, —CH═CH₂, —C≡CH, Br, I, F,bis-(4-ethynyl-benzyl)-amino, dipropargylamino, di-hex-5-ynyl-amino,di-pent-4-ynyl-amino, di-but-3-ynyl-amino, propargyloxy, hex-5-ynyloxy,pent-4-ynyloxy, di-but-3-ynyloxy, 2-hex-5-ynyloxy-ethyldisulfanyl,2-pent-4-ynyloxy-ethyldisulfanyl, 2-but-3-ynyloxy-ethyldisulfanyl,2-propargyloxy-ethyldisulfanyl, bis-benzyloxy-methyl,[1,3]dioxolan-2-yl, and [1,3]dioxan-2-yl.

In other embofiments, the R³ moiety is an aryl group substituted with asuitably protected amino group. According to another aspect, the R³moiety is phenyl substituted with a suitably protected amino group.

In other embodiments, the R³ moiety of the R¹ group of formula II is aprotected hydroxyl group. In certain embodiments the protected hydroxylof the R³ moiety is an ester, carbonate, sulfonate, allyl ether, ether,silyl ether, alkyl ether, arylalkyl ether, or alkoxyalkyl ether. Incertain embodiments, the ester is a formate, acetate, proprionate,pentanoate, crotonate, or benzoate. Exemplary esters include formate,benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate,4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate(trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate,p-benylbenzoate, 2,4,6-trimethylbenzoate. Exemplary carbonates include9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl,2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonate.Examples of suitable silyl ethers include trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl ether, andother trialkylsilyl ethers. Exemplary alkyl ethers include methyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allylether, or derivatives thereof. Exemplary alkoxyalkyl ethers includeacetals such as methoxymethyl, methylthiomethyl,(2-methoxyethoxy)methyl, benzyloxymethyl,beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyran-2-yl ether.Exemplary arylalkyl ethers include benzyl, p-methoxybenzyl (MPM),3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl ethers.

In certain embodiments, the R³ moiety of the R¹ group of formula II is amono-protected or di-protected amino group. In certain embodiments R³ isa mono-protected amine. In certain embodiments R³ is a mono-protectedamine selected from aralkylamines, carbamates, ally! amines, or amides.Exemplary mono-protected amino moieties include t-butyloxycarbonylamino,ethyloxycarbonylamino, methyloxycarbonylamino,trichloroethyloxy-carbonylamino, allyloxycarbonylamino,benzyloxocarbonylamino, allylamino, benzylamino,fluorenylmethylcarbonyl, formamido, acetamido, chloroacetamido,dichloroacetamido, trichloroacetamido, phenylacetamido,trifluoroacetamido, benzamido, and t-butyldiphenylsilylamino. In otherembodiments R³ is a di-protected amine. Exemplary di-protected aminesinclude di-benzylamine, di-allylamine, phthalimide, maleimide,succinimide, pyrrole, 2,2,5,5-tetramethyl-[1,2,5]azadisilolidine, andazide. In certain embodiments, the R³ moiety is phthalimido. In otherembodiments, the R³ moiety is mono- or di-benzylamino or mono- ordi-allylamino. In certain embodiments, the R¹ group is2-dibenzylaminoethoxy.

In other embodiments, the R³ moiety of the R¹ group of formula II is aprotected aldehyde group. In certain embodiments the protected aldehydomoiety of R³ is an acyclic acetal, a cyclic acetal, a hydrazone, or animine. Exemplary R³ groups include dimethyl acetal, diethyl acetal,diisopropyl acetal, dibenzyl acetal, bis(2-nitrobenzyl) acetal,1,3-dioxane, 1,3-dioxolane, and semicarbazone. In certain embodiments,R³ is an acyclic acetal or a cyclic acetal. In other embodiments, R³ isa dibenzyl acetal.

In yet other embodiments, the R³ moiety of the R¹ group of formula II isa protected carboxylic acid group. In certain embodiments, the protectedcarboxylic acid moiety of R³ is an optionally substituted ester selectedfrom C₁₋₆ aliphatic or aryl, or a silyl ester, an activated ester, anamide, or a hydrazide. Examples of such ester groups include methyl,ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, and phenyl ester. Inother embodiments, the protected carboxylic acid moiety of R³ is anoxazoline or an ortho ester. Examples of such protected carboxylic acidmoieties include oxazolin-2-yl and 2-methoxy-[1,3]dioxin-2-yl. Incertain embodiments, the R¹ group is oxazolin-2-ylmethoxy or2-oxazolin-2-yl-1-propoxy.

According to another embodiment, the R³ moiety of the R¹ group offormula II is a protected thiol group. In certain embodiments, theprotected thiol of R³ is a disulfide, thioether, silyl thioether,thioester, thiocarbonate, or a thiocarbamate. Examples of such protectedthiols include triisopropylsilyl thioether, t-butyldimethylsilylthioether, t-butyl thioether, benzyl thioether, p-methylbenzylthioether, triphenylmethyl thioether, and p-methoxyphenyldiphenylmethylthioether. In other embodiments, R³ is an optionally substitutedthioether selected from alkyl, benzyl, or triphenylmethyl, ortrichloroethoxycarbonyl thioester. In certain embodmients, R³ is—S—S-pyridin-2-yl, —S—SBn, —S—SCH₃, or —S—S(p-ethynylbenzyl). In otherembodmients, R³ is —S—S-pyridin-2-yl. In still other embodiments, the R¹group is 2-triphenylmethylsulfanyl-ethoxy.

In certain embodiments, the R³ moiety of the R¹ group of formula II is acrown ether. Examples of such crown ethers include 12-crown-4,15-crown-5, and 18-crown-6.

In still other embodiments, the R³ moiety of the R¹ group of formula IIis a detectable moiety. According to one aspect of the invention, the R³moiety of the R¹ group of formula II is a fluorescent moiety. Suchfluorescent moieties are well known in the art and include coumarins,quinolones, benzoisoquinolones, hostasol, and Rhodamine dyes, to namebut a few. Exemplary fluorescent moieties of the R³ group of R¹ includeanthracen-9-yl, pyren-4-yl, 9-H-carbazol-9-yl, the carboxylate ofrhodamine B, and the carboxylate of coumarin 343.

In certain embodiments, the R³ moiety of the R¹ group of formula II is agroup suitable for Click chemistry. Click reactions tend to involvehigh-energy (“spring-loaded”) reagents with well-defined reactioncoordinates, giving rise to selective bond-forming events of wide scope.Examples include the nucleophilic trapping of strained-ringelectrophiles (epoxide, aziridines, aziridinium ions, episulfoniumions), certain forms of carbonyl reactivity (aldehydes and hydrazines orhydroxylamines, for example), and several types of cycloadditionreactions. The azide-alkyne 1,3-dipolar cycloaddition is one suchreaction. Click chemistry is known in the art and one of ordinary skillin the art would recognize that certain R³ moieties of the presentinvention are suitable for Click chemistry.

In certain embodiments, the R³ moiety of the R¹ group of formula II is agroup suitable for Click chemistry. Click reactions tend to involvehigh-energy (“spring-loaded”) reagents with well-defined reactioncoordinates, giving rise to selective bond-forming events of wide scope.Examples include the nucleophilic trapping of strained-ringelectrophiles (epoxide, aziridines, aziridinium ions, episulfoniumions), certain forms of carbonyl reactivity (aldehydes and hydrazines orhydroxylamines, for example), and several types of cycloadditionreactions. The azide-alkyne 1,3-dipolar cycloaddition is one suchreaction. Click chemistry is known in the art and one of ordinary skillin the art would recognize that certain R³ moieties of the presentinvention are suitable for Click chemistry.

Compounds of formula II having R³ moieties suitable for Click chemistryare useful for conjugating said compounds to biological systems ormacromolecules such as proteins, viruses, and cells, to name but a few.The Click reaction is known to proceed quickly and selectively underphysiological conditions. In contrast, most conjugation reactions arecarried out using the primary amine functionality on proteins (e.g.lysine or protein end-group). Because most proteins contain a multitudeof lysines and arginines, such conjugation occurs uncontrollably atmultiple sites on the protein. This is particularly problematic whenlysines or arginines are located around the active site of an enzyme orother biomolecule. Thus, another embodiment of the present inventionprovides a method of conjugating the R¹ groups of a compound of formulaII to a macromolecule via Click chemistry. Yet another embodiment of thepresent invention provides a macromolecule conjugated to a compound offormula II via the R¹ group.

According to one embodiment, the R³ moiety of the R¹ group of formula IIis an azide-containing group. According to another embodiment, the R³moiety of the R¹ group of formula II is an alkyne-containing group. Incertain embodiments, the R³ moiety of the R¹ group of formula II has aterminal alkyne moiety. In other embodiments, R³ moiety of the R¹ groupof formula II is an alkyne moiety having an electron withdrawing group.Accordingly, in such embodiments, the R³ moiety of the R¹ group offormula II is

wherein E is an electron withdrawing group and y is 0-6. Such electronwithdrawing groups are known to one of ordinary skill in the art. Incertain embodiments. E is an ester. In other embodiments, the R³ moietyof the R¹ group of formula II is

wherein E is an electron withdrawing group, such as a —C(O)O— group andy is 0-6.

As defined generally above, the Q group of formula II is a valence bondor a bivalent, saturated or unsaturated, straight or branched C₁₋₁₂alkylene chain, wherein 0-6 methylene units of Q are independentlyreplaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO₂—,—NHSO₂—, —SO₂NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or —NHC(O)O—, wherein-Cy- is an optionally substituted 5-8 membered bivalent, saturated,partially unsaturated, or aryl ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, or an optionally substituted8-10 membered bivalent saturated, partially unsaturated, or arylbicyclic ring having 0-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, Q is a valencebond. In other embodiments, Q is a bivalent, saturated C₁₋₁₂ alkylenechain, wherein 0-6 methylene units of Q are independently replaced by-Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—, or —C(O)—, wherein -Cy- is anoptionally substituted 5-8 membered bivalent, saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, or an optionally substituted 8-10membered bivalent saturated, partially unsaturated, or aryl bicyclicring having 0-5 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

In certain embodiments, Q is -Cy- (i.e. a C₁ alkylene chain wherein themethylene unit is replaced by -Cy-), wherein -Cy- is an optionallysubstituted 5-8 membered bivalent, saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. According to one aspect of the present invention,-Cy- is an optionally substituted bivalent aryl group. According toanother aspect of the present invention, -Cy- is an optionallysubstituted bivalent phenyl group. In other embodiments, -Cy- is anoptionally substituted 5-8 membered bivalent, saturated carbocyclicring. In still other embodiments, -Cy- is an optionally substituted 5-8membered bivalent, saturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. Exemplary -Cy-groups include bivalent rings selected from phenyl, pyridyl,pyrimidinyl, cyclohexyl, cyclopentyl, or cyclopropyl.

In certain embodiments, the R^(x) group of formula II is a crosslinkableamino acid side-chain group and R^(y) is a hydrophobic amino acidside-chain group. Such hydrophilic, or crosslinkable, amino acidside-chain groups include tyrosine, serine, cysteine, threonine,aspartic acid (also known as aspartate, when charged), glutamic acid(also known as glutamate, when charged), asparagine, histidine, lysine,arginine, and glutamine. Such hydrophobic amino acid side-chain groupsinclude a suitably protected tyrosine side-chain, a suitably protectedserine side-chain, a suitably protected threonine side-chain,phenylalanine, alanine, valine, leucine, tryptophan, proline, benzyl andalkyl glutamates, or benzyl and alkyl aspartates or mixtures thereof.Such ionic amino acid side chain groups includes a lysine side-chain,arginine side-chain, or a suitably protected lysine or arginineside-chain, an aspartic acid side chain, glutamic acid side-chain, asuitably protected aspartic acid or glutamic acid side-chain, histidineor a suitably protected histidine side-chain. One of ordinary skill inthe art would recognize that protection of a polar or hydrophilic aminoacid side-chain can render that amino acid nonpolar. For example, asuitably protected tyrosine hydroxyl group can render that tyrosinenonpolar and hydrophobic by virtue of protecting the hydroxyl group.Suitable protecting groups for the hydroxyl, amino, and thiol, andcarboylate functional groups of R^(x) and R^(y) are as described herein.

In other embodiments, the R^(y) group of formula II comprises a mixtureof hydrophobic and hydrophilic amino acid side-chain groups such thatthe overall poly(amino acid) block comprising R^(y) is hydrophobic. Suchmixtures of amino acid side-chain groups include phenylalanine/tyrosine,phenalanine/serine, leucine/tyrosine, and the like. According to anotherembodiment, R^(y) is a hydrophobic amino acid side-chain group selectedfrom phenylalanine, alanine, or leucine, and one or more of tyrosine,serine, or threonine.

As defined above, R^(x) is a natural or unnatural amino acid side-chaingroup capable of forming cross-links. It will be appreciated that avariety of amino acid side-chain functional groups are capable of suchcross-linking, including, but not limited to, carboxylate, hydroxyl,thiol, and amino groups. Examples of R^(x) moieties having functionalgroups capable of forming cross-links include a glutamic acidside-chain, —CH₂C(O)CH, an aspartic acid side-chain, —CH₂CH₂C(O)OH, acystein side-chain, —CH₂SH, a serine side-chain, —CH₂OH, an aldehydecontaining side-chain, —CH₂C(O)H, a lysine side-chain, —(CH₂)₄NH₂, anarginine side-chain, —(CH₂)₃NHC(═NH)NH₂, a histidine side-chain,—CH₂-imidazol-4-yl.

In other embodiments, R^(x) comprises a mixture of hydrophilic aminoacid side-chain groups. Such mixtures of amino acid side-chain groupsinclude those having a carboxylic acid functionality, a hydroxylfunctionality, a thiol functionality, and/or amine functionality. Itwill be appreciated that when R^(x) comprises a mixture of hydrophilicamino acid side-chain functionalities, then multiple crosslinking canoccur. For example, when R^(x) comprises a carboxylic acid-containingside-chain (e.g., aspartic acid or glutamic acid) and a thiol-containingside-chain (e.g., cysteine), then the amino acid block can have bothzinc crosslinking and cysteine crosslinking (dithiol). This sort ofmixed crosslinked block is advantageous for the delivery of therapeuticdrugs to the cytosol of diseased cells. When R^(x) comprises anamine-containing side-chain (e.g., lysine or arginine) and athiol-containing side-chain (e.g., cysteine), then the amino acid blockcan have both imine (e.g. Schiff base) crosslinking and cysteinecrosslinking (dithiol). The zinc and ester crosslinked carboxylic acidfunctionality and the imine (e.g. Schiff base) crosslinked aminefunctionality are reversible in acidic organelles (i.e. endosomes,lysosome) while disulfides are reduced in the cytosol by glutathione orother reducing agents resulting in drug release exclusively in thecytoplasm.

As defined generally above, the R^(2a) group of formula II is amono-protected amine, a di-protected amine, —NHR⁴, —N(R⁴)₂, —NHC(O)R⁴,—NR⁴C(O)R⁴, —NHC(O)NHR⁴, —NHC(O)N(R⁴)₂, —NR⁴C(O)NHR⁴, —NR⁴C(O)N(R⁴)₂,—NHC(O)OR⁴, —NR⁴C(O)OR⁴, —NHSO₂R⁴; or —NR⁴SO₂R⁴, wherein each R⁴ isindependently an optionally substituted group selected from aliphatic, a5-8 membered saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, an8-10-membered saturated, partially unsaturated, or aryl bicyclic ringhaving 0-5 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or a detectable moiety, or two R⁴ on the same nitrogen atom aretaken together with said nitrogen atom to form an optionally substituted4-7 membered saturated, partially unsaturated, or aryl ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, the R^(2a) group of formula II is —NHR⁴ or—N(R⁴)₂ wherein each R⁴ is an optionally substituted aliphatic group.One exemplary R⁴ group is 5-norboren-2-yl-methyl. According to yetanother aspect of the present invention, the R^(2a) group of formula IIis —NHR⁴ wherein R⁴ is a C₁₋₆ aliphatic group substituted with N₃.Examples include —CH₂N₃. In some embodiments, R⁴ is an optionallysubstituted C₁₋₆ alkyl group. Examples include methyl, ethyl, propyl,butyl, pentyl, hexyl, 2-(tetrahydropyran-2-yloxy)ethyl,pyridin-2-yldisulfanylmethyl, methyldisulfanylmethyl,(4-acetylenylphenyl)methyl, 3-(methoxycarbonyl)-prop-2-ynyl,methoxycarbonylmethyl,2-(N-methyl-N-(4-acetylenylphenyl)carbonylamino)-ethyl,2-phthalimidoethyl, 4-bromobenzyl, 4-chlorobenzyl, 4-fluorobenzyl,4-iodobenzyl, 4-propargyloxybenzyl, 2-nitrobenzyl,4-(bis-4-acetylenylbenzyl)aminomethyl-benzyl, 4-propargyloxy-benzyl,4-dipropargylamino-benzyl, 4-(2-propargyloxy-ethyldisulfanyl)benzyl,2-propargyloxy-ethyl, 2-propargyldisulfanyl-ethyl, 4-propargyloxy-butyl,2-(N-methyl-N-propargylamino)ethyl, and2-(2-dipropargylaminoethoxy)-ethyl. In other embodiments, R⁴ is anoptionally substituted C₂₋₆ alkenyl group. Examples include vinyl,allyl, crotyl, 2-propenyl, and but-3-enyl. When R⁴ group is asubstituted aliphatic group, suitable substituents on R⁴ include N₃, CN,and halogen. In certain embodiments, R⁴ is —CH₂CN, —CH₂CH₂CN,—CH₂CH(OCH₃)_(2∝, 4)-(bisbenzyloxymethyl)phenylmethyl, and the like.

According to another aspect of the present invention, the R^(2a) groupof formula II is —NHR⁴ wherein R⁴ is an optionally substituted C₂₋₆alkynyl group. Examples include —CC≡CH, —CH₂C≡CH, —CH₂C≡CCH₃, and—CH₂CH₂C≡CH.

In certain embodiments, the R^(2a) group of formula II is —NHR⁴ whereinR⁴ is an optionally substituted 5-8-membered aryl ring. In certainembodiments, R⁴ is optionally substituted phenyl or optionallysubstituted pyridyl. Examples include phenyl,4-t-butoxycarbonylaminophenyl, 4-azidomethylphenyl,4-propargyloxyphenyl, 2-pyridyl, 3-pyridyl, and 4-pyridyl. In certainembodiments, R^(2a) is 4-t-butoxycarbonylaminophenylamino,4-azidomethylphenamino, or 4-propargyloxyphenylamino.

In certain embodiments, the R^(2a) group of formula II is —NHR⁴ whereinR⁴ is an optionally substituted phenyl ring. Suitable substituents onthe R⁴ phenyl ring include halogen; —(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘); —NO₂; —CN;—N₃; —(CH₂)₀₋₄N(R^(∘) ₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘); —N(R^(∘)C(S)R^(∘);—(CH₂)₀₋₄N(R^(∘)C(O)NR^(∘) ₂; —N(R⁶⁰² )C(S)NR^(∘) ₂;—(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘)C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘) ₂; —C(S)NR^(∘) ₂;—(CH₂)₀₋₄OC(O)NR^(∘) ₂; —C(O)N(OR^(∘))R^(∘); —C(O)C(O)R⁶⁰² ;—C(O)CH₂C(O)R^(∘); —(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘); —(CH₂₀₋₄S(O)₂R^(∘); —(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘); —S(O)₂NR^(∘)₂; —(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘))S(O)₂R^(∘);—N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘)₂; SiR^(∘) ₃; wherein each independent occurrence of R^(∘) is as definedherein supra. In other embodiments, the R^(2a) group of formula II is-NHR⁴ wherein R⁴ is phenyl substituted with one or more optionallysubstituted C₁₋₆aliphatic groups. In still other embodiments, R⁴ isphenyl substituted with vinyl, allyl, acetylenyl, —CH₂N₃, —CH₂CH₂N₃,—CH₂C≡CCH₃, or —CH₂C≡CH.

In certain embodiments, the R^(2a) group of formula II is —NHR⁴ whereinR⁴ is phenyl substituted with N₃, N(R^(∘) ₂, CO₂R^(∘), or C(O)R° whereineach R^(∘) is independently as defined herein supra.

In certain embodiments, the R^(2a) group of formula II is —N(R⁴)₂wherein each R⁴ is independently an optionally substituted groupselected from aliphatic, phenyl, naphthyl, a 5-6 membered aryl ringhaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or a 8-10 membered bicyclic aryl ring having 1-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or a detectablemoiety.

In other embodiments, the R^(2a) group of formula II is —N(R⁴)₂ whereinthe two R⁴ groups are taken together with said nitrogen atom to form anoptionally substituted 4-7 membered saturated, partially unsaturated, oraryl ring having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. According to another embodiment, the two R⁴ groupsare taken together to form a 5-6-membered saturated or partiallyunsaturated ring having one nitrogen wherein said ring is substitutedwith one or two oxo groups. Such R^(2a) groups include, but are notlimited to, phthalimide, maleimide and succinimide.

In certain embodiments, the R^(2a) group of formula II is amono-protected or di-protected amino group. In certain embodimentsR^(2a) is a mono-protected amine. In certain embodiments R^(2a) is amono-protected amine selected from aralkylamines, carbamates, allylamines, or amides. Exemplary mono-protected amino moieties includet-butyloxycarbonylamino, ethyloxycarbonylamino, methyloxycarbonylamino,trichloroethyloxy-carbonylamino, allyloxycarbonylamino,benzyloxocarbonylamino, allylamino, benzylamino,fluorenylmethylcarbonyl, formamido, acetamido, chloroacetamido,dichloroacetamido, trichloroacetamido, phenylacetamido,trifluoroacetamido, benzamido, and t-butyldiphenylsilylamino. In otherembodiments R^(2a) is a di-protected amine. Exemplary di-protected aminomoieties include di-benzylamino, di-allylamino, phthalimide, maleimido,succinimido, pyrrolo, 2,2,5,5-tetramethyl-[1,2,5]azadisilolidino, andazido. In certain embodiments, the R^(2a) moiety is phthalimido. Inother embodiments, the R^(2a) moiety is mono- or di-benzylamino or mono-or di-allylamino.

In certain embodiments, the R^(2a) group of formula II comprises a groupsuitable for Click chemistry. One of ordinary skill in the art wouldrecognize that certain R^(2a) groups of the present invention aresuitable for Click chemistry.

Compounds of formula II having R^(2a) groups comprising groups suitablefor Click chemistry are useful for conjugating said compounds tobiological systems such as proteins, viruses, and cells, to name but afew. After conjugation to a biomolecule, drug, cell, substrate, or thelike, the other end-group functionality, corresponding to the R¹ moietyof formula II, can be used to attach targeting groups for cell specificdelivery including, but not limited to, fluorescent dyes, covalentattachment to surfaces, and incorporation into hydrogels. Thus, anotherembodiment of the present invention provides a method of conjugating theR^(2a) group of a compound of formula II to a macromolecule via Clickchemistry. Yet another embodiment of the present invention provides amacromolecule conjugated to a compound of formula II via the R^(2a)group.

According to one embodiment, the R^(2a) group of formula II is anazide-containing group. According to another embodiment, the R^(2a)group of formula II is an alkyne-containing group.

In certain embodiments, the R^(2a) group of formula II has a terminalalkyne moiety. In other embodiments, the R^(2a) group of formula II isan alkyne-containing moiety having an electron withdrawing group.Accordingly, in such embodiments, the R^(2a) group of formula II is

wherein E is an electron withdrawing group and y is 0-6. Such electronwithdrawing groups are known to one of ordinary skill in the art. Incertain embodiments, E is an ester. In other embodiments, the R^(2a)group of formula II is

wherein E is an electron withdrawing group, such as a —C(O)O— group andy is 0-6.

Exemplary compounds of the present invention are set forth in Tables 1to 4, below. Table 1 sets forth exemplary compounds of the formula:

wherein each w is 25-1000, each x is 1-50, each y is 1-50, each z is1-100, p is the sum of y and z, and each dotted bond represents thepoint of attachment to the rest of the molecule.

TABLE 1 Compound A¹ A² A³ E¹ E² 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

Table 2 sets forth exemplary compounds of the formula:

wherein each x is 100-500, each y is 4-20, each z is 5-50, and eachdotted bond represents the point of attachment to the rest of themolecule.

TABLE 2 Compound A¹ A² E¹ E² 99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

Table 3 sets forth exemplary compounds of the formula:

wherein each v is 100-500, each w is 4-20, x is 4-20, each y is 5-50,each z is 5-50, p is the sum of y and z, and each dotted bond representsthe point of attachment to the rest of the molecule.

TABLE 3 Compound A¹ A² A³ A⁴ E¹ E² 193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

Table 4 sets forth exemplary compounds of the formula:

wherein each w is 25-1000, each x is 1-50, y is 1-50, each z is 1-100,and each dotted bond represents the point of attachment to the rest ofthe molecule.

TABLE 4 Compound A¹ A² A³ E¹ E² 213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

B. Crosslinking Chemistries

In addition to advances in polymer micelle technology, significantefforts have been made in the development of stimuli-responsivepolymeric materials that can respond to environmental pH changes. SeeChatterjee, J.; Haik, Y.; Chen, C. J. J. App. Polym. Sci. 2004, 91,3337-3341; Du, J. Z.; Armes, S. P. J. Am. Chem. Soc. 2005, 127,12800-12801; and Twaites, B. R.; de las Heras Alarcon, C.; Cunliffe, D.;Lavigne, M.; Pennadam, S.; Smith, J. R.; Gorecki, D. C.; Alexander, C.J. Control. Release 2004, 97, 551-566. This is of importance forsensitive protein and nucleic acid-based drugs where escape from acidicintracellular compartments (i.e. endosome and lysosome) and cytoplasmicrelease are required to achieve therapeutic value. See Murthy, N.;Campbell, J.; Fausto, N.; Hoffman, A. S.; Stayton, P. S. J. Control.Release 2003, 89, 365-374; El-Sayed, M. E. H.; Hoffman, A. S.; Stayton,P. S. J. Control. Release 2005, 104, 417-427; and Liu, Y.; Wenning, L.;Lynch, M.; Reineke, T. J. Am. Chem. Soc. 2004, 126, 7422-7423.Acid-sensitive delivery systems that can successfully escape theendosome and transport small-molecule chemotherapeutic drugs into thecytoplasm are also of interest since these carriers can bypass many ofthe cellular mechanisms responsible for multi-drug resistance. In someof these cases, the polymers are designed to respond to the significantpH gradient between the blood (pH 7.4) and the late-early endosome (pH˜5.0-6.0).

There is additional interest in developing the cancer-specific,pH-sensitive targeting of therapeutics. For example, rapidly growingcells found in solid tumors have elevated glycolytic rates and increasedlactic acid production when compared to healthy cells. These factors,along with poor lymphatic drainage present in cancerous tissue result inan excess of lactic acid and a subtle pH gradient between the blood andthe solid tumor microenvironment (pH 6.5-7.0). See Kalllinowski, F.;Schlenger, K. H.; Runkel, S.; Kloes, M.; Stohrer, M.; Okunieff, P.;Vaupel, P. Cancer Res. 1989, 49, 3759-3764. Although the design ofmaterials which can respond to such small pH variations is clearlychallenging, this mechanism, coupled with the EPR effect, represent aneffective method for limiting drug release to solid tumors.

In certain embodiments, the amphiphilic block copolymers andcell-responsive polymer micelles of the present invention are designedto combine the concepts of crosslinked polymer micelles and pH-sensitivedrug targeting to construct “smart” nanovectors that are infinitelystable to dilution in the bloodstream but are chemically programmed torelease their therapeutic payload in response to pH changes commonlyfound in solid tumors and cancer cells. By utilizing cancer-responsivenanovectors in conjunction with potent chemotherapeutic agents,long-standing clinical problems such as post-injection micelle stabilityand the targeted delivery of encapsulated therapeutics to cancer cellsare addressed. Unlike previous examples of micelle crosslinking (i.e.,core and shell crosslinking), the multi-block approach of the presentinvention allows for the effective crosslinking of polymer segmentslocated at the interface of the hydrophobic and hydrophilic polymerblocks as shown in FIG. 1. This approach is advantageous because stablemicelles are prepared without sacrificing loading efficiency or alteringthe drug molecule during core crosslinking.

In contrast to shell-crosslinked micelles, the crosslinking ofmultiblock copolymer micelles in accordance with the present inventionis accomplished without large dilution volumes because micelle-micellecoupling does not occur. Such crosslinking will enhancepost-administration circulation time leading to more efficient passivedrug targeting by the EPR effect and improved active targeting usingcancer-specific targeting groups. In addition, stimuli-responsivecrosslinking may offer another targeting mechanism to isolate therelease of the chemotherapy drug exclusively within the tumor tissue andcancer cell cytoplasm.

Crosslinking reactions designed for drug delivery preferably meet acertain set of requirements to be deemed safe and useful for in vivoapplications. For example, in other embodiments, the crosslinkingreaction would utilize non-cytotoxic reagents, would be insensitive towater, would not alter the drug to be delivered, and in the case ofcancer therapy, would be reversible at pH levels commonly encountered intumor tissue (pH ˜6.8) or acidic organelles in cancer cells (pH˜5.0-6.0).

In certain embodiments, micelles of the present invention comprise acrosslinked multiblock polymer of formula III:

wherein:

n is 10-2500;

m is 1 to 1000;

m′ is 1 to 1000;

L is a bivalent, saturated or unsaturated, straight or branched C₁₋₁₂alkylene chain, wherein 0-6 methylene units of L are independentlyreplaced by -M-, -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—,—SO₂—, —NHSO₂—, —SO₂NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or —NHC(O)O—,wherein:

-   -   -M- is a suitable bivalent metal;    -   -Cy- is an optionally substituted 5-8 membered bivalent,        saturated, partially unsaturated, or aryl ring having 0-4        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, or an optionally substituted 8-10 membered bivalent        saturated, partially unsaturated, or aryl bicyclic ring having        0-5 heteroatoms independently selected from nitrogen, oxygen, or        sulfur;

R^(y) is a hydrophobic or ionic, natural or unnatural amino acidside-chain group;

R¹ is —Z(CH₂CH₂Y)_(p)(CH₂)_(t)R³, wherein:

-   -   Z is —O—, —S—, —C≡C—, or —CH₂—;    -   each Y is independently —O— or —S—;    -   p is 0-10;    -   t is 0-10; and    -   R³ is —N₃, —CN, a mono-protected amine, a di-protected amine, a        protected aldehyde, a protected hydroxyl, a protected carboxylic        acid, a protected thiol, a 9-30 membered crown ether, or an        optionally substituted group selected from aliphatic, a 5-8        membered saturated, partially unsaturated, or aryl ring having        0-4 heteroatoms independently selected from nitrogen, oxygen, or        sulfur, an 8-10 membered saturated, partially unsaturated, or        aryl bicyclic ring having 0-5 heteroatoms independently selected        from nitrogen, oxygen, or sulfur, or a detectable moiety;    -   Q is a valence bond or a bivalent, saturated or unsaturated,        straight or branched C₁₋₁₂ alkylene chain, wherein 0-6 methylene        units of Q are independently replaced by -Cy-, —O—, —NH—, —S—,        —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO₂—, —NHSO₂—, —SO₂NH—,        —NHC(O)—, —C(O)NH—, —OC(O)NH—, or —NHC(O)O—, wherein:        -   -Cy- is an optionally substituted 5-8 membered bivalent,            saturated, partially unsaturated, or aryl ring having 0-4            heteroatoms independently selected from nitrogen, oxygen, or            sulfur, or an optionally substituted 8-10 membered bivalent            saturated, partially unsaturated, or aryl bicyclic ring            having 0-5 heteroatoms independently selected from nitrogen,            oxygen, or sulfur;    -   R^(2a) is a mono-protected amine, a di-protected amine, —N(R⁴)₂,        —NR⁴C(O)R⁴, —NR⁴C(O)N(R⁴)₂, —NR⁴C(O)OR⁴, or —NR⁴SO₂R⁴; and    -   each R⁴ is independently an optionally substituted group        selected from hydrogen, aliphatic, a 5-8 membered saturated,        partially unsaturated, or aryl ring having 0-4 heteroatoms        independently selected from nitrogen, oxygen, or sulfur, an 8-10        membered saturated, partially unsaturated, or aryl bicyclic ring        having 0-5 heteroatoms independently selected from nitrogen,        oxygen, or sulfur, or a detectable moiety, or:        -   two R⁴ on the same nitrogen atom are taken together with            said nitrogen atom to form an optionally substituted 4-7            membered saturated, partially unsaturated, or aryl ring            having 1-4 heteroatoms independently selected from nitrogen,            oxygen, or sulfur.

According to another embodiment, the present invention providescompounds of formula III, as described above, wherein said compoundshave a polydispersity index (“PDI”) of about 1.0 to about 1.2. Accordingto another embodiment, the present invention provides compounds offormula III, as described above, wherein said compound has apolydispersity index (“PDI”) of about 1.03 to about 1.15. According toyet another embodiment, the present invention provides compounds offormula I, as described above, wherein said compound has apolydispersity index (“PDI”) of about 1.10 to about 1.20. According toother embodiments, the present invention provides compounds of formulaIII having a PDI of less than about 1.10.

As defined generally above, the n group of formula III is 10-2500. Incertain embodiments, the present invention provides compounds of formulaIII, as described above, wherein n is about 225. In other embodiments, nis about 10 to about 40. In other embodiments, n is about 40 to about60. In still other embodiments, n is about 90 to about 150. In stillother embodiments, n is about 200 to about 250. In other embodiments, nis about 300 to about 375. In other embodiments, n is about 400 to about500. In still other embodiments, n is about 650 to about 750.

In certain embodiments, the m′ group of formula III is about 5 to about500. In certain embodiments, the m′ group of formula III is about 10 toabout 250. In other embodiments, m′ is about 10 to about 50. In otherembodiments, m′ is about 20 to about 40. According to yet anotherembodiment, m′ is about 50 to about 75. According to other embodiments,m and m′ are independently about 10 to about 100. In certainembodiments, m is 5-50. In other embodiments, m is 5-10. In otherembodiments, m is 10-20. In certain embodiments, m and m′ add up toabout 30 to about 60. In still other embodiments, m is 1-20 repeat unitsand m′ is 10-50 repeat units.

As defined generally above, the L group of formula III is a bivalent,saturated or unsaturated, straight or branched C₁₋₁₂ alkylene chain,wherein 0-6 methylene units of L are independently replaced by -M-, Cy ,—O—, NH—, —S—, —C(O)—, —SO—, —SO₂—, NHC(O)—, C(O)NH—, OC(O)NH—, or—NHC(O)O—, wherein -M- is a suitable bivalent metal, and -Cy- is anoptionally substituted 5-8 membered bivalent, saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, or an optionally substituted 8-10membered bivalent saturated, partially unsaturated, or aryl bicyclicring having 0-5 heteroatoms independently selected from nitrogen,oxygen, or sulfur. It will be appreciated that the L group of formulaIII represents crosslinked amino acid side-chain groups. In certainembodiments, the crosslinked amino acid side-chain groups correspond tothe R^(x) moiety of compounds of formulae I and II as described herein.In certain embodiments, the L group of formula III represents a metalcrosslinked amino acid side-chain group, a hydrazone crosslinked aminoacid side-chain group, an ester crosslinked amino acid side-chain group,an amide crosslinked side-chain group, an imine (e.g. Schiff base)crosslinked side-chain group, or a disulfide crosslinked side-chaingroup.

In certain embodiments, the L group of formula III comprises -M-. Inother embodiments, -M- is zinc, calcium, iron or aluminum. In yet otherembodiments, -M- is strontium, manganese, palladium, silver, gold,cadmium, chromium, indium, or lead.

In other embodiments, the L group of formula III is a bivalent,saturated or unsaturated, straight or branched C₁₋₁₂ alkylene chainwherein 2 methylene units of L are independently replaced by —C(O)—,—C(O)NH—, —NHC(O)—, —S—, —C(O)O—, —OC(O)—, —C(O)NHN—, —═NNHC(O)—, —═N—,—N═—, -M-OC(O)—, or —C(O)O-M-. According to another embodiment, the Lgroup of formula III is a bivalent, saturated or unsaturated, straightor branched C₁₋₆ alkylene chain, wherein two methylene units of L arereplaced by —C(O)— or —C(O)NH—. In other embodiments, the L group offormula III is a bivalent, saturated or unsaturated, straight orbranched C₁₋₁₂ alkylene chain having at least 2 units of unsaturation.According to yet another embodiment, the L group of formula III is abivalent, saturated or unsaturated, straight or branched C₁₋₁₂ alkylenechain wherein two methylene units of L are replaced by —NH—. Accordingto yet another embodiment, the L group of formula III is a bivalent,saturated or unsaturated, straight or branched C₁₋₁₂ alkylene chainwherein two methylene units of L are replaced by —C(O)NHN.

In certain embodiments, the -M- moiety of the L group of formula III iszinc. In other embodiments, L forms a zinc-dicarboxylate crosslinkingmoiety. In certain embodiments, the crosslinking utilizes zinc-mediatedcoupling of carboxylic acids, a highly selective and pH-sensitivereaction that is performed in water. This reaction, which is widely usedin cough lozenge applications, involves the association of zinc ionswith carboxylic acids at basic pH. See Bakar, N. K. A.; Taylor, D. M.;Williams, D. R. Chem. Spec. Bioavail. 1999, 11, 95-101; and Eby, G. A.J. Antimicrob. Chemo. 1997, 40, 483-493. These zinc-carboxylate bondsreadily dissociate in the presence of acid.

Scheme 1 above illustrates the reaction of an aqueous zinc ion (e.g.from zinc chloride) with two equivalents of an appropriate carboxylicacid to form the zinc dicarboxylate. This reaction occurs rapidly andirreversibly in a slightly basic pH environment but upon acidification,is reversible within a tunable range of pH 4.0-6.8 to reform ZnX₂, whereX is the conjugate base. One of ordinary skill in the art will recognizethat a variety of natural and unnatural amino acid side-chains have acarboxylic acid moiety that can be crosslinked by zinc or anothersuitable metal.

In certain embodiments, L represents aspartic acid side-chainscrosslinked with zinc. Without wishing to be bound by theory, it isbelieved that the zinc aspartate crosslinks are stable in the bloodcompartment (pH 7.4), allowing for effective accumulation of thedrug-loaded micelles in solid tumors by passive and active targetingmechanisms. In the presence of lactic acid concentrations commonlyencountered in solid tumors or in acidic organelles of cancer cells,rapid degradation of the metal crosslinks leading to micelledissociation and release of the drug at the tumor site. Preliminary,qualitative studies have shown that crosslinked zinc aspartate segmentsare reversible in the presence of α-hydroxyacids.

The choice of zinc as a crosslinking metal is advantageous for effectivemicelle crosslinking. Zinc chloride and the zinc lactate by-product aregenerally recognized as non-toxic, and other safety concerns are notanticipated. Pharmaceutical grade zinc chloride is commonly used inmouthwash and as a chlorophyll stabilizer in vegetables while zinclactate is used as an additive in toothpaste and drug preparation. Thereaction is reversible within a tunable pH range, selective towardcarboxylic acids, and should not alter the encapsulated chemotherapyagents. While zinc has been chosen as an exemplary metal for micellecrosslinking, it should be noted that many other metals undergo acidsensitive coupling with carboxylic acids. These metals include calcium,iron and aluminum, to name but a few. One or more of these metals can besubstituted for zinc.

The ultimate goal of metal-mediated crosslinking is to ensure micellestability when diluted in the blood (pH 7.4) followed by rapiddissolution and drug release in response to a finite pH change such asthose found in cancer cells. Previous reports suggest a widely variableand tunable dissociation pH for zinc-acid bonds (from approximately 2:0to 7.0) depending on the carboxylic acid used and number of bondsformed. See Cannan, R. K.; Kibrick, A. J. Am. Chem. Soc. 1938, 60,2314-2320. Without wishing to be bound by theory, it is believed thatthe concentration of zinc chloride and the number of aspartic acid, orother carboxylic acid-containing amino acid, repeat units in thecrosslinking block will ultimately control the pH at which completemicelle disassembly occurs. The synthetic versatility of the blockcopolymer design is advantageous since one or more variables are tunedto achieve the desired pH reversibility. By simple adjustment of zincchloride/polymer stoichiometry, pH-reversible crosslinking is finelytuned across the pH range of interest. For example, higher zincconcentrations yield more zinc crosslinks which require higher acidconcentrations (i.e. lower pH) to dissociate. Adjustments inzinc/polymer stoichiometry will yield the desired pH reversibility,however other variables such as increasing the poly(aspartic acid) blocklength (i.e. 15-25 repeat units) further tune the reversiblecrosslinking reaction if necessary.

In other embodiments, L comprises a mixture of crosslinked hydrophilicamino acid side-chain groups. Such mixtures of amino acid side-chaingroups include those having a carboxylic acid functionality, a hydroxylfunctionality, a thiol functionality, and/or amine functionality. Itwill be appreciated that when L comprises a mixture of crosslinkedhydrophilic amino acid side-chain functionalities, then multiplecrosslinking can occur. For example, when L comprises a carboxylicacid-containing side-chain (e.g., aspartic acid or glutamic acid) and athiol-containing side-chain (e.g., cysteine), then the amino acid blockcan have both zinc crosslinking and cysteine crosslinking (dithiol).This sort of mixed crosslinked block is advantageous for the delivery oftherapeutic drugs to the cytosol of diseased cells because a secondstimuli must be present to allow for drug release. For example, micellespossessing both carboxylic acid-zinc crosslinking and cysteine dithiolcrosslinking would be required to enter an acidic environment (e.g. atumor) and enter an environment with a high concentration of glutathione(e.g. in the cell cytoplasm). When L comprises an amine-containingside-chain (e.g., lysine or arginine) and a thiol-containing side-chain(e.g., cysteine), then the amino acid block can have both imine (e.g.Schiff base) crosslinking and cysteine crosslinking (dithiol). The zincand ester crosslinked carboxylic acid functionality and the imine (e.g.Schiff base) crosslinked amine functionality are reversible in acidicorganelles (i.e. endosomes, lysosome) while disulfides are reduced inthe cytosol by glutathione or other reducing agents resulting in drugrelease exclusively in the cytoplasm.

Exemplary crosslinking reactions and resulting L groups are depicted inFIGS. 2 through 10.

In certain embodiments, the R³ moiety of the R¹ group of formula III is—N₃.

In other embodiments, the R³ moiety of the R¹ group of formula III is—CN.

In still other embodiments, the R³ moiety of the R¹ group of formula IIIis a mono-protected amine or a di-protected amine.

In certain embodiments, the R³ moiety of the R¹ group of formula III isan optionally substituted aliphatic group. Examples include t-butyl,5-norbornene-2-yl, octane-5-yl, acetylenyl, trimethylsilylacetylenyl,triisopropylsilylacetylenyl, and t-butyldimethylsilylacetylenyl. In someembodiments, said R³ moiety is an optionally substituted alkyl group. Inother embodiments, said R³ moiety is an optionally substituted alkynylor alkenyl group. When said R³ moiety is a substituted aliphatic group,suitable substituents on R³ include CN, N₃, trimethylsilyl,triisopropylsilyl, t-butyldimethylsilyl, N-methyl propiolamido,N-methyl-4-acetylenylanilino, N-methyl-4-acetylenylbenzoamido,bis-(4-ethynyl-benzyl)-amino, dipropargylamino, di-hex-5-ynyl-amino,di-pent-4-ynyl-amino, di-but-3-ynyl-amino, propargyloxy, hex-5-ynyloxy,pent-4-ynyloxy, di-but-3-ynyloxy, N-methyl-propargylamino,N-methyl-hex-5-ynyl-amino, N-methyl-pent-4-ynyl-amino,N-methyl-but-3-ynyl-amino, 2-hex-5-ynyldisulfanyl,2-pent-4-ynyldisulfanyl, 2-but-3-ynyldisulfanyl, and2-propargyldisulfanyl. In certain embodiments, the R¹ group is2-(N-methyl-N-(ethynylcarbonyl)amino)ethoxy, 4-ethynylbenzyloxy, or2-(4-ethynylphenoxy)ethoxy.

In certain embodiments, the R³ moiety of the R¹ group of formula III isan optionally substituted aryl group. Examples include optionallysubstituted phenyl and optionally substituted pyridyl. When said R³moiety is a substituted aryl group, suitable substituents on R³ includeCN, N₃, NO₂, —CH₃, —CH₂N₃, —CH═CH₂, Br, I, F,bis-(4-ethynyl-benzyl)-amino, dipropargylamino, di-hex-5-ynyl-amino,di-pent-4-ynyl-amino, di-but-3-ynyl-amino, propargyloxy, hex-5-ynyloxy,pent-4-ynyloxy, di-but-3-ynyloxy, 2-hex-5-ynyloxy-ethyldisulfanyl,2-pent-4-ynyloxy-ethyldisulfanyl, 2-but-3-ynyloxy-ethyldisulfanyl,2-propargyloxy-ethyldisulfanyl, bis-benzyloxy-methyl,[1,3]dioxolan-2-yl, and [1,3]dioxan-2-yl.

In other embodiments, the R³ moiety is an aryl group substituted with asuitably protected amino group. According to another aspect, the R³moiety is phenyl substituted with a suitably protected amino group.

In other embodiments, the R³ moiety of the R¹ group of formula III is aprotected hydroxyl group. In certain embodiments the protected hydroxylof the R³ moiety is an ester, carbonate, sulfonate, allyl ether, ether,silyl ether, alkyl ether, arylalkyl ether, or alkoxyalkyl ether. Incertain embodiments, the ester is a formate, acetate, proprionate,pentanoate, crotonate, or benzoate. Exemplary esters include formate,benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate,4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate(trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate,p-benylbenzoate, 2,4,6-trimethylbenzoate. Exemplary carbonates include9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl,2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonate.Examples of suitable silyl ethers include trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl ether, andother trialkylsilyl ethers. Exemplary alkyl ethers include methyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allylether, or derivatives thereof. Exemplary alkoxyalkyl ethers includeacetals such as methoxymethyl, methylthiomethyl,(2-methoxyethoxy)methyl, benzyloxymethyl,beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyran-2-yl ether.Exemplary arylalkyl ethers include benzyl, p-methoxybenzyl (MPM),3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl ethers.

In certain embodiments, the R³ moiety of the R¹ group of formula III isa mono-protected or di-protected amino group. In certain embodiments R³is a mono-protected amine. In certain embodiments R³ is a mono-protectedamine selected from aralkylamines, carbamates, allyl amines, or amides.Exemplary mono-protected amino moieties include t-butyloxycarbonylamino,ethyloxycarbonylamino, methyloxycarbonylamino,trichloroethyloxy-carbonylamino, allyloxycarbonylamino,benzyloxocarbonylamino, allylamino, benzylamino,fluorenylmethylcarbonyl, formamido, acetamido, chloroacetamido,dichloroacetamido, trichloroacetamido, phenylacetamido,trifluoroacetamido, benzamido, and t-butyldiphenylsilylamino. In otherembodiments R³ is a di-protected amine. Exemplary di-protected aminesinclude di-benzylamine, di-allylamine, phthalimide, maleimide,succinimide, pyrrole, 2,2,5,5-tetramethyl-[1,2,5]azadisilolidine, andazide. In certain embodiments, the R³ moiety is phthalimido. In otherembodiments, the R³ moiety is mono- or di-benzylamino or mono- ordi-allylamino. In certain embodiments, the R¹ group is2-dibenzylaminoethoxy.

In other embodiments, the R³ moiety of the R¹ group of formula I is aprotected aldehyde group. In certain embodiments the protected aldehydomoiety of R³ is an acyclic acetal, a cyclic acetal, a hydrazone, or animine. Exemplary R³ groups include dimethyl acetal, diethyl acetal,diisopropyl acetal, dibenzyl acetal, bis(2-nitrobenzyl) acetal,1,3-dioxane, 1,3-dioxolane, and semicarbazone. In certain embodiments,R³ is an acyclic acetal or a cyclic acetal. In other embodiments, R³ isa dibenzyl acetal.

In yet other embodiments, the R³ moiety of the R^(I) group of formulaIII is a protected carboxylic acid group. In certain embodiments, theprotected carboxylic acid moiety of R³ is an optionally substitutedester selected from C₁₋₆ aliphatic or aryl, or a silyl ester, anactivated ester, an amide, or a hydrazide. Examples of such ester groupsinclude methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, andphenyl ester. In other embodiments, the protected carboxylic acid moietyof R³ is an oxazoline or an ortho ester. Examples of such protectedcarboxylic acid moieties include oxazolin-2-yl and2-methoxy-[1,3]dioxin-2-yl. In certain embodiments, the R¹ group isoxazolin-2-ylmethoxy or 2-oxazolin-2-yl-1-propoxy.

According to another embodiments, the R³ moiety of the R¹ group offormula III is a protected thiol group. In certain embodiments, theprotected thiol of R³ is a disulfide, thioether, silyl thioether,thioester, thiocarbonate, or a thiocarbamate. Examples of such protectedthiols include triisopropylsilyl thioether, t-butyldimethylsilylthioether, t-butyl thioether, benzyl thioether, p-methylbenzylthioether, triphenylmethyl thioether, and p-methoxyphenyldiphenylmethylthioether. In other embodiments, R³ is an optionally substitutedthioether selected from alkyl, benzyl, or triphenylmethyl, ortrichloroethoxycarbonyl thioester. In certain embodiments, R³ is—S—S-pyridin-2-yl, —S—SBn, —S—SCH₃, or —S—S(p-ethynylbenzyl). In otherembodiments, R³ is —S—S-pyridin-2-yl. In still other embodiments, the R¹group is 2-triphenylmethyl sulfanyl-ethoxy.

In certain embodiments, the R³ moiety of the R¹ group of formula III isa crown ether. Examples of such crown ethers include 12-crown-4,15-crown-5, and 18-crown-6.

In still other embodiments, the R³ moiety of the R¹ group of formula IIIis a detectable moiety. According to one aspect of the invention, the R³moiety of the R¹ group of formula III is a fluorescent moiety. Suchfluorescent moieties are well known in the art and include coumarins,quinolones, benzoisoquinolones, hostasol, and Rhodamine dyes, to namebut a few. Exemplary fluorescent moieties of the R³ group of R¹ includeanthracen-9-yl, pyren-4-yl, 9-H-carbazol-9-yl, the carboxylate ofrhodamine B, and the carboxylate of coumarin 343.

In certain embodiments, the R³ moiety of the R¹ group of formula III isa group suitable for Click chemistry. Click reactions tend to involvehigh-energy (“spring-loaded”) reagents with well-defined reactioncoordinates, giving rise to selective bond-forming events of wide scope.Examples include the nucleophilic trapping of strained-ringelectrophiles (epoxide, aziridines, aziridinium ions, episulfoniumions), certain forms of carbonyl reactivity (aldehydes and hydrazines orhydroxylamines, for example), and several types of cycloadditionreactions. The azide-alkyne 1,3-dipolar cycloaddition is one suchreaction. Click chemistry is known in the art and one of ordinary skillin the art would recognize that certain R³ moieties of the presentinvention are suitable for Click chemistry.

In certain embodiments, the R³ moiety of the R¹ group of formula III isa group suitable for Click chemistry. Click reactions tend to involvehigh-energy (“spring-loaded”) reagents with well-defined reactioncoordinates, giving rise to selective bond-forming events of wide scope.Examples include the nucleophilic trapping of strained-ringelectrophiles (epoxide, aziridines, aziridinium ions, episulfoniumions), certain forms of carbonyl reactivity (aldehydes and hydrazines orhydroxylamines, for example), and several types of cycloadditionreactions. The azide-alkyne 1,3-dipolar cycloaddition is one suchreaction. Click chemistry is known in the art and one of ordinary skillin the art would recognize that certain R³ moieties of the presentinvention are suitable for Click chemistry.

Compounds of formula III having R³ moieties suitable for Click chemistryare useful for conjugating said compounds to biological systems ormacromolecules such as proteins, viruses, and cells, to name but a few.The Click reaction is known to proceed quickly and selectively underphysiological conditions. In contrast, most conjugation reactions arecarried out using the primary amine functionality on proteins (e.g.lysine or protein end-group). Because most proteins contain a multitudeof lysines and arginines, such conjugation occurs uncontrollably atmultiple sites on the protein. This is particularly problematic whenlysines or arginines are located around the active site of an enzyme orother biomolecule. Thus, another embodiment of the present inventionprovides a method of conjugating the R¹ groups of a compound of formulaIII to a macromolecule via Click chemistry. Yet another embodiment ofthe present invention provides a macromolecule conjugated to a compoundof formula III via the R¹ group.

According to one embodiment, the R³ moiety of the R¹ group of formulaIII is an azide-containing group. According to another embodiment, theR³ moiety of the R¹ group of formula III is an alkyne-containing group.In certain embodiments, the R³ moiety of the R¹ group of formula III hasa terminal alkyne moiety. In other embodiments, R³ moiety of the R¹group of formula III is an alkyne moiety having an electron withdrawinggroup. Accordingly, in such embodiments, the R³ moiety of the R¹ groupof formula III is

wherein E is an electron withdrawing group and y is 0-6. Such electronwithdrawing groups are known to one of ordinary skill in the art. Incertain embodiments. E is an ester. In other embodiments, the R³ moietyof the R¹ group of formula III

is wherein E is an electron withdrawing group, such as a —C(O)O— groupand y is 0-6.

As defined generally above, Q is a valence bond or a bivalent, saturatedor unsaturated, straight or branched C₁₋₁₂ alkylene chain, wherein 0-6methylene units of Q are independently replaced by -Cy-, —O—, —NH—, —S—,—OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO₂—, —NHSO₂—, —SO₂NH—, —NHC(O)—,—C(O)NH—, —OC(O)NH—, or —NHC(O)O—, wherein -Cy- is an optionallysubstituted 5-8 membered bivalent, saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or an optionally substituted 8-10 membered bivalentsaturated, partially unsaturated, or aryl bicyclic ring having 0-5heteroatoms independently selected from nitrogen, oxygen, or sulfur. Incertain embodiments, Q is a valence bond. In other embodiments, Q is abivalent, saturated C₁₋₁₂ alkylene chain, wherein 0-6 methylene units ofQ are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—,or —C(O)—, wherein -Cy- is an optionally substituted 5-8 memberedbivalent, saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, oran optionally substituted 8-10 membered bivalent saturated, partiallyunsaturated, or aryl bicyclic ring having 0-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, Q is -Cy- (i.e. a C₁ alkylene chain wherein themethylene unit is replaced by -Cy-), wherein -Cy- is an optionallysubstituted 5-8 membered bivalent, saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. According to one aspect of the present invention,-Cy- is an optionally substituted bivalent aryl group. According toanother aspect of the present invention, -Cy- is an optionallysubstituted bivalent phenyl group. In other embodiments, -Cy- is anoptionally substituted 5-8 membered bivalent, saturated carbocyclicring. In still other embodiments, -Cy- is an optionally substituted 5-8membered bivalent, saturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. Exemplary -Cy-groups include bivalent rings selected from phenyl, pyridyl,pyrimidinyl, cyclohexyl, cyclopentyl, or cyclopropyl.

In certain embodiments, R^(y) is a hydrophobic amino acid side-chaingroup. Such hydrophobic amino acid side-chain groups include a suitablyprotected tyrosine side-chain, a suitably protected serine side-chain, asuitably protected threonine side-chain, phenylalanine, alanine, valine,leucine, tryptophan, proline, benzyl and alkyl glutamates, or benzyl andalkyl aspartates or mixtures thereof Such ionic amino acid side chaingroups includes a lysine side-chain, arginine side-chain, or a suitablyprotected lysine or arginine side-chain, an aspartic acid side chain,glutamic acid side-chain, or a suitably protected aspartic acid orglutamic acid side-chain. One of ordinary skill in the art wouldrecognize that protection of a polar or hydrophilic amino acidside-chain can render that amino acid nonpolar. For example, a suitablyprotected tyrosine hydroxyl group can render that tyrosine nonpolar andhydrophobic by virtue of protecting the hydroxyl group. Suitableprotecting groups for the hydroxyl, amino, and thiol functional groupsof R^(y) are as described herein.

In other embodiments, R^(y) comprises a mixture of hydrophobic andhydrophilic amino acid side-chain groups such that the overallpoly(amino acid) block comprising R^(y) is hydrophobic. Such mixtures ofamino acid side-chain groups include phenylalanine/tyrosine,phenalanine/serine, leucine/tyrosine, and the like. According to anotherembodiment, R^(y) is a hydrophobic amino acid side-chain group selectedfrom phenylalanine, alanine, or leucine, and one or more of tyrosine,serine, or threonine.

As defined generally above, the R^(2a) group of formula III is amono-protected amine, a di-protected amine, —NHR⁴, —N(R⁴)₂, —NHC(O)R⁴,—NR⁴C(O)R⁴, —NHC(O)NHR⁴, —NHC(O)N(R⁴)₂, —NR⁴C(O)NHR⁴, —NR⁴C(O)N(R⁴)₂,—NHC(O)OR⁴, or —NR⁴SO₂R⁴, wherein each R⁴ is independently an optionallysubstituted group selected from aliphatic, a 5-8 membered saturated,partially unsaturated, or aryl ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, an 8-10-membered saturated,partially unsaturated, or aryl bicyclic ring having 0-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or a detectablemoiety, or two R⁴ on the same nitrogen atom are taken together with saidnitrogen atom to form an optionally substituted 4-7 membered saturated,partially unsaturated, or aryl ring having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, the R^(2a) group of formula III is —NHR⁴ or—N(R⁴)₂ wherein each R⁴ is an optionally substituted aliphatic group.One exemplary R⁴ group is 5-norbornen-2-yl-methyl. According to yetanother aspect of the present invention, the R^(2a) group of formula IIIis —NHR⁴ wherein R⁴ is a C₁₋₆ aliphatic group substituted with N₃.Examples include —CH₂N₃. In some embodiments, R⁴ is an optionallysubstituted C₁₋₆ alkyl group. Examples include methyl, ethyl, propyl,butyl, pentyl, hexyl, 2-(tetrahydropyran-2-yloxy)ethyl,pyridin-2-yldisulfanylmethyl, methyldisulfanylmethyl,(4-acetylenylphenyl)methyl, 3-(methoxycarbonyl)-prop-2-ynyl,methoxycarbonylmethyl,2-(N-methyl-N-(4-acetylenylphenyl)carbonylamino)-ethyl,2-phthalimidoethyl, 4-bromobenzyl, 4-chlorobenzyl, 4-fluorobenzyl,4-iodobenzyl, 4-propargyloxybenzyl, 2-nitrobenzyl,4-(bis-4-acetylenylbenzyl)aminomethyl-benzyl, 4-propargyloxy-benzyl,4-dipropargylamino-benzyl, 4-(2-propargyloxy-ethyldisulfanyl)benzyl,2-propargyloxy-ethyl, 2-propargyldisulfanyl-ethyl, 4-propargyloxy-butyl,2-(N-methyl-N-propargylamino)ethyl, and2-(2-dipropargylaminoethoxy)-ethyl. In other embodiments, R⁴ is anoptionally substituted C₂₋₆ alkenyl group. Examples include vinyl,allyl, crotyl, 2-propenyl, and but-3-enyl. When R⁴ group is asubstituted aliphatic group, suitable substituents on R⁴ include N₃, CN,and halogen. In certain embodiments, R⁴ is —CH₂CN, —CH₂CH₂CN,—CH₂CH(OCH₃)₂, 4-(bisbenzyloxymethyl)phenylmethyl, and the like.

According to another aspect of the present invention, the R^(2a) groupof formula III is —NHR⁴ wherein R⁴ is an optionally substituted C₂₋₆alkynyl group. Examples include —CC≡CH, —CH₂C≡CH, —CH₂C≡CCH₃, and—CH₂CH₂C≡CH,

In certain embodiments, the R^(2a) group of formula III is —NHR⁴ whereinR⁴ is an optionally substituted 5-8-membered aryl ring. In certainembodiments, R⁴ is optionally substituted phenyl or optionallysubstituted pyridyl. Examples include phenyl,4-t-butoxycarbonylaminophenyl, 4-azidomethylphenyl,4-propargyloxyphenyl, 2-pyridyl, 3-pyridyl, and 4-pyridyl. In certainembodiments, R^(2a) is 4-t-butoxycarbonylaminophenylamino,4-azidomethylphenamino, or 4-propargyloxyphenylamino.

In certain embodiments, the R^(2a) group of formula III is —NHR⁴ whereinR⁴ is an optionally substituted phenyl ring. Suitable substituents onthe R⁴ phenyl ring include halogen; —(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘);—(CH₂)₀₋₄CH(OR^(∘) ₂; —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘; —NO) ₂; —CN;—N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘); —N(R^(∘))C(S)R^(∘);—(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘) ₂;—(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄-C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘) ₂; —C(S)NR^(∘) ₂;—(CH₂)₀₋₄OC(O)NR^(∘) ₂; —C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘);—C(O)CH₂C(O)R^(∘); —C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘);—(CH₂)₀₋₄S(O)₂R^(∘), —(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘);—S(O)₂NR^(∘) ₂; —(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂;—N(R^(∘))S(O)₂R^(∘); —N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘);—P(O)R^(∘) ₂; —OP(O)R^(∘) ₂; SiR^(∘) ₃, wherein each independentoccurrence of R^(∘) is as defined herein supra. In other embodiments,the R^(2a) group of formula III is —NHR⁴ wherein R⁴ is phenylsubstituted with one or more optionally substituted C₁₋₆ aliphaticgroups. In still other embodiments, R⁴ is phenyl substituted with vinyl,allyl, acetylenyl, —CH₂N₃, —CH₂CH₂N₃, —CH₂C≡CCH₃, or —CH₂C≡CH.

In certain embodiments, the R^(2a) group of formula III is —NHR⁴ whereinR⁴ is phenyl substituted with N₃, N(R^(∘)) ₂, CO₂R^(∘), or C(O)R^(∘)wherein each R^(∘) is independently as defined herein supra.

In certain embodiments, the R^(2a) group of formula III is —N(R⁴)₂wherein each R⁴ is independently an optionally substituted groupselected from aliphatic, phenyl, naphthyl, a 5-6 membered aryl ringhaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or a 8-10 membered bicyclic aryl ring having 1-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or a detectablemoiety.

In other embodiments, the R^(2a) group of formula III is —N(R⁴)₂ whereinthe two R⁴ groups are taken together with said nitrogen atom to form anoptionally substituted 4-7 membered saturated, partially unsaturated, oraryl ring having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. According to another embodiment, the two R⁴ groupsare taken together to form a 5-6-membered saturated or partiallyunsaturated ring having one nitrogen wherein said ring is substitutedwith one or two oxo groups. Such R^(2a) groups include, but are notlimited to, phthalimide, maleimide and succinimide.

In certain embodiments, the R^(2a) group of formula III is amono-protected or di-protected amino group. In certain embodimentsR^(2a) is a mono-protected amine. In certain embodiments R^(2a) is amono-protected amine selected from aralkylamines, carbamates, allylamines, or amides. Exemplary mono-protected amino moieties includet-butyloxycarbonylamino, ethyloxycarbonylamino, methyloxycarbonylamino,trichloroethyloxy-carbonylamino, allyloxycarbonylamino,benzyloxocarbonylamino, allylamino, benzylamino,fluorenylmethylcarbonyl, formamido, acetamido, chloroacetamido,dichloroacetamido, trichloroacetamido, phenylacetamido,trifluoroacetamido, benzamido, and t-butyldiphenylsilylamino. In otherembodiments R^(2a) is a di-protected amine. Exemplary di-protected aminomoieties include di-benzylamino, di-allylamino, phthalimide, maleimido,succinimido, pyrrolo, 2,2,5,5-tetramethyl-[1,2,5]azadisilolidino, andazido. In certain embodiments, the R^(2a) moiety is phthalimido. Inother embodiments, the R^(2a) moiety is mono- or di-benzylamino or mono-or di-allylamino.

In certain embodiments, the R^(2a) group of formula III comprises agroup suitable for Click chemistry. One of ordinary skill in the artwould recognize that certain R^(2a) groups of the present invention aresuitable for Click chemistry.

Compounds of formula III having R^(2a) groups comprising groups suitablefor Click chemistry are useful for conjugating said compounds tobiological systems such as proteins, viruses, and cells, to name but afew. After conjugation to a biomolecule, drug, cell, substrate, or thelike, the other end-group functionality, corresponding to the R¹ moietyof formula III, can be used to attach targeting groups for cell specificdelivery including, but not limited to, fluorescent dyes, covalentattachment to surfaces, and incorporation into hydrogels. Thus, anotherembodiment of the present invention provides a method of conjugating theR^(2a) group of a compound of formula III to a macromolecule via Clickchemistry. Yet another embodiment of the present invention provides amacromolecule conjugated to a compound of formula III via the R^(2a)group.

According to one embodiment, the R^(2a) group of formula III is anazide-containing group. According to another embodiment, the R^(2a)group of formula III is an alkyne-containing group.

In certain embodiments, the R^(2a) group of formula III has a terminalalkyne moiety. In other embodiments, the R^(2a) group of formula III isan alkyne-containing moiety having an electron withdrawing group.Accordingly, in such embodiments, the R^(2a) group of formula III is

wherein E is an electron withdrawing group and y is 0-6. Such electronwithdrawing groups are known to one of ordinary skill in the art. Incertain embodiments, E is an ester. In other embodiments, the R^(2a)group of formula III is

wherein E is an electron withdrawing group, such as a —C(O)O— group andy is 0-6.

Exemplary R¹ groups of any of formulae I, II, and III are set forth inTable 5, below.

TABLE 5 Representative R¹ Groups

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

q

r

s

t

u

v

w

x

y

z

aa

bb

cc

dd

ee

ff

gg

hh

ii

jj

kk

ll

mm

nn

oo

pp

qq

rr

ss

tt

uu

vv

ww

xx

yy

zz

aaa

bbb

ccc

ddd

eee

fff

ggg

hhh

iii

jjj

kkk

lll

mmm

nnn

ooo

ppp

qqq

rrr

sss

ttt

uuu

vvv

www

xxx

yyy

zzz

One of ordinary skill in the art would recognize that certain R¹ groupsdepicted in Table 5 are protected groups, e.g. protected amine,protected hydroxyl, protected thiol, protected carboxylic acid, orprotected alkyne groups. Each of these protected groups is readilydeprotected (see, for example, Green). Accordingly, the deprotectedgroups corresponding to the protected groups set forth in Table 5 arealso contemplated. According to another embodiment, the R¹ group of anyof formulae I, II, and III is selected from a deprotected group of Table5.

Additional exemplary R¹ groups of any of formulae I, II, and III are setforth in Table 5a, below.

TABLE 5a Representative R¹ Groups

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

q

r

s

t

u

v

w

x

y

z

aa

bb

cc

dd

ee

ff

gg

hh

ii

jj

kk

ll

mm

nn

oo

pp

qq

rr

ss

tt

uu

vv

ww

xx

yy

zz

aaa

bbb

ccc

ddd

eee

fff

ggg

hhh

iii

jjj

kkk

lll

mmm

nnn

ooo

ppp

qqq

rrr

sss

ttt

In certain embodiments, the R¹ group of any of formulae I, II, and IIIis selected from any of those R¹ groups depicted in Table 5, supra. Inother embodiments, the R¹ group of any of formulae I, II, and III isgroup k or l. In yet other embodiments, the R¹ group of any of formulaeI, II, and III is n, o, cc, dd, ee, ff, hh, h, ii, jj, ll, or uu. Instill other embodiments, the R¹ group of any of formulae I, II, and IIIis h, aa, yy, zz, or aaa.

According to another aspect of the present invention, the R¹ group ofany of formulae I, II, and III is q, r, s, t, www, xxx, or yyy.

In other embodiments, the R¹ group of any of formulae I, II, and III isselected from any of those R¹ groups depicted in Tables 1-4, supra.

Exemplary R^(2a) groups of any of formulae I, II, and III are set forthin Table 6, below.

TABLE 6 Representative R^(2a) Groups

i

ii

iii

iv

v

vi

vii

viii

ix

x

x

xi

xii

xiii

xiv

xv

xvi

xvii

xviii

xix

xx

xxi

xxii

xxiii

xxiv

xxv

xxvi

xxvii

xxviii

xxix

xxx

xxxi

xxxii

xxxiii

xxxiv

xxxv

xxxvi

xxxvii

xxxviii

xxxix

xl

xli

xlii

xliii

xliv

xlv

xlvi

xlvii

In certain embodiments, the R^(2a) group of any of formulae I, II, andIII is selected from any of those R^(2a) groups depicted in Table 6,supra. In other embodiments, the R^(2a) group of any of formulae I, II,and III is group v, viii, xvi, xix, xxii, xxx, xxxi, xxxii, xxxiii,xxxiv, xxxv, xxxvi, xxxvii, or xlii. In yet other embodiments, theR^(2a) group of any of formulae I, II, and III is xv, xviii, xx, xxi,xxxviii, or xxxix. In certain embodiments, the R^(2a) group of any offormulae I, II, and III is xxxiv.

According to another embodiment, the R^(2a) group of any of formulae I,II, and III is selected from any of those R^(2a) groups depicted inTables 1-4, supra.

One of ordinary skill in the art would recognize that certain R^(2a)groups depicted in Table 6 are protected groups, e.g. protected amine,protected hydroxyl, protected thiol, protected carboxylic acid, orprotected alkyne groups. Each of these protected groups is readilydeprotected (see, for example, Green). Accordingly, the deprotectedgroups corresponding to the protected groups set forth in Table 6 arealso contemplated. According to another embodiment, the R^(2a) group ofany of formulae I, II, and III is selected from a deprotected group ofTable 6.

C. Drug Loading

As described generally above, in certain embodiments the presentinvention provides a drug-loaded micelle comprising a multiblockcopolymer which comprises a polymeric hydrophilic block, a crosslinkedpoly(amino acid block), and a poly(amino acid block), characterized inthat said micelle has a drug-loaded inner core, a crosslinked outercore, and a hydrophilic shell. As described herein, micelles of thepresent invention can be loaded with any hydrophobic or ionictherapeutic agent.

According to another embodiment, the present invention provides adrug-loaded micelle comprising a multiblock copolymer of formula I:

wherein:

n is 10-2500;

m is 1 to 1000;

m′ is 1 to 1000;

R^(x) is a natural or unnatural amino acid side-chain group that iscapable of crosslinking;

R^(y) is a hydrophobic or ionic, natural or unnatural amino acidside-chain group;

R¹ is —Z(CH₂CH₂Y)_(p)(CH₂)_(t)R³, wherein:

-   -   Z is —O—, —S—, —C≡C—, or —CH₂—;    -   each Y is independently —O— or —S—;    -   p is 0-10;    -   t is 0-10; and    -   R³ is —N₃, —CN, a mono-protected amine, a di-protected amine, a        protected aldehyde, a protected hydroxyl, a protected carboxylic        acid, a protected thiol, a 9-30 membered crown ether, or an        optionally substituted group selected from aliphatic, a 5-8        membered saturated, partially unsaturated, or aryl ring having        0-4 heteroatoms independently selected from nitrogen, oxygen, or        sulfur, an 8-10 membered saturated, partially unsaturated, or        aryl bicyclic ring having 0-5 heteroatoms independently selected        from nitrogen, oxygen, or sulfur, or a detectable moiety;

Q is a valence bond or a bivalent, saturated or unsaturated, straight orbranched C₁₋₁₂ alkylene chain, wherein 0-6 methylene units of Q areindependently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—,—C(O)—, —SO—, —SO₂—, —NHSO₂—, —SO₂NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or—NHC(O)O—, wherein:

-   -   -Cy- is an optionally substituted 5-8 membered bivalent,        saturated, partially unsaturated, or aryl ring having 0-4        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, or an optionally substituted 8-10 membered bivalent        saturated, partially unsaturated, or aryl bicyclic ring having        0-5 heteroatoms independently selected from nitrogen, oxygen, or        sulfur;

R^(2a) is a mono-protected amine, a di-protected amine, —N(R⁴)₂,—NR⁴C(O)R⁴, —NR⁴C(O)N(R⁴)₂, —NR⁴C(O)OR⁴, or —NR⁴SO₂R⁴; and

each R⁴ is independently an optionally substituted group selected fromhydrogen, aliphatic, a 5-8 membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, oraryl bicyclic ring having 0-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or a detectable moiety, or:

-   -   two R⁴ on the same nitrogen atom are taken together with said        nitrogen atom to form an optionally substituted 4-7 membered        saturated, partially unsaturated, or aryl ring having 1-4        heteroatoms independently selected from nitrogen, oxygen, or        sulfur.

Embodiments with respect to each of the R¹, R^(2a), Q, R^(x), R^(y), n,m, and m′ groups of formula I, are as described in various classes andsubclasses, both singly and in combination, herein.

In certain embodiments, R^(x) is a crosslinkable amino acid side-chaingroup and R^(y) is a hydrophobic amino acid side-chain group. Suchhydrophilic, or crosslinkable, amino acid side-chain groups includetyrosine, serine, cysteine, threonine, aspartic acid (also known asaspartate, when charged), glutamic acid (also known as glutamate, whencharged), asparagine, and glutamine. Such hydrophobic amino acidside-chain groups include a suitably protected tyrosine side-chain, asuitably protected serine side-chain, a suitably protected threonineside-chain, phenylalanine, alanine, valine, leucine, tryptophan,proline, benzyl and alkyl glutamates, or benzyl and alkyl aspartates ormixtures thereof. Such ionic amino acid side chain groups includes alysine side-chain, arginine side-chain, or a suitably protected lysineor arginine side-chain, an aspartic acid side chain, glutamic acidside-chain, or a suitably protected aspartic acid or glutamic acidside-chain. One of ordinary skill in the art would recognize thatprotection of a polar or hydrophilic amino acid side-chain can renderthat amino acid nonpolar. For example, a suitably protected tyrosinehydroxyl group can render that tyrosine nonpolar and hydrophobic byvirtue of protecting the hydroxyl group. Suitable protecting groups forthe hydroxyl, amino, and thiol, and carboylate functional groups ofR^(x) and R^(y) are as described herein.

In other embodiments, R^(y) comprises a mixture of hydrophobic andhydrophilic amino acid side-chain groups such that the overallpoly(amino acid) block comprising R^(y) is hydrophobic. Such mixtures ofamino acid side-chain groups include phenylalanine/tyrosine,phenalanine/serine, leucine/tyrosine, and the like. According to anotherembodiment, R^(y) is a hydrophobic amino acid side-chain group selectedfrom phenylalanine, alanine, or leucine, and one or more of tyrosine,serine, or threonine.

As defined above, R^(x) is a natural or unnatural amino acid side-chaingroup capable of forming cross-links. It will be appreciated that avariety of amino acid side-chain functional groups are capable of suchcross-linking, including, but not limited to, carboxylate, hydroxyl,thiol, and amino groups. Examples of R^(x) moieties having functionalgroups capable of forming cross-links include a glutamic acidside-chain, —CH₂C(O)CH, an aspartic acid side-chain, —CH₂CH₂C(O)OH, acystein side-chain, —CH₂SH, a serine side-chain, —CH₂OH, an aldehydecontaining side-chain, —CH₂C(O)H, a lysine side-chain, —(CH₂)₄NH₂, anarginine side-chain, —(CH₂)₃NHC(═NH)NH₂, a histidine side-chain,—CH₂-imidazol-4-yl.

As defined generally above, the R^(2a) group of formula I is amono-protected amine, a di-protected amine, —NHR⁴, —N(R⁴)₂, —NHC(O)R⁴,—NR⁴C(O)R⁴, —NHC(O)NHR⁴, —NHC(O)N(R⁴)₂, —NR⁴C(O)NHR⁴, —NR4C(O)N(R⁴)₂,—NHC(O)OR⁴, —NR⁴C(O)OR⁴, —NHSO₂R⁴, or —NR⁴SO₂R⁴, wherein each R⁴ isindependently an optionally substituted group selected from aliphatic, a5-8 membered saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, an8-10-membered saturated, partially unsaturated, or aryl bicyclic ringhaving 0-5 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or a detectable moiety, or two R⁴ on the same nitrogen atom aretaken together with said nitrogen atom to form an optionally substituted4-7 membered saturated, partially unsaturated, or aryl ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur.

One of ordinary skill in the art will recognize that the R^(2a) moietycan interact with the encapsulated drug. In certain embodiments, theR^(2a) moiety is hydrophobic when the encapsulated drug is hydrophobic.Such hydrophobic R^(2a) groups include linear and branched alkanes. Inother embodiments, the R^(2a) moiety is ionic when the encapsulated drugis ionic. Such ionic R^(2a) groups include alkyl amines when theencapsulated drug is a cationic therapeutic (i.e. DNA and RNAtherapeutics, oligopeptide and protein therapeutics). Other ionic R^(2a)groups include alkyl carboxylic, sulfonic, and phosponic acids when theencapsulated drug is an anionic therapeutic (i.e. oligopeptide andprotein therapeutics).

The accomodation of structurally diverse therapeutic agents within amicelle of the present invention is effected by adjusting the poly(aminoacid) block, i.e., the block comprising R^(y). For example, when R^(y)is a hydrophobic natural or unnatural amino acid side-chain, micelles ofthe present invention are useful for encapsulating hydrophobictherapeutic agents.

In certain embodiments, micelles of the present invention are loadedwith a hydrophobic drug. In accordance with such embodiments, R^(y) is anatural or unnatural hydrophobic amino acid side-chain group. Suchhydrophobic amino acid side-chain groups include a suitably protectedtyrosine side-chain, a suitably protected serine side-chain, a suitablyprotected threonine side-chain, phenylalanine, alanine, valine, leucine,tryptophan, proline, benzyl and alkyl glutamates, or benzyl and alkylaspartates, or mixtures thereof. One of ordinary skill in the art wouldrecognize that protection of a polar or hydrophilic amino acidside-chain can render that amino acid nonpolar. For example, a suitablyprotected tyrosine hydroxyl group can render that tyrosine nonpolar andhydrophobic by virtue of protecting the hydroxyl group. Suitableprotecting groups for the hydroxyl, amino, and thiol, and carboxylatefunctional groups of R^(y) are as described herein.

In other embodiments, the R^(y) group of formula I comprises a mixtureof hydrophobic and hydrophilic amino acid side-chain groups such thatthe overall poly(amino acid) block comprising R^(y) is hydrophobic. Suchmixtures of amino acid side-chain groups include phenylalanine/tyrosine,phenalanine/serine, leucine/tyrosine, and the like. According to anotherembodiment, R^(y) is a hydrophobic amino acid side-chain group selectedfrom phenylalanine, alanine, or leucine, and one or more of tyrosine,serine, or threonine.

Hydrophobic small molecule drugs suitable for loading into micelles ofthe present invention are well known in the art. In certain embodiments,the present invention provides a drug-loaded micelle as describedherein, wherein the drug is a hydrophobic drug selected from thosedescribed herein, infra.

In other embodiments, when the R^(y) group of formula I is an ionicnatural or unnatural amino acid side-chain, micelles of the presentinvention are useful for encapsulating ionic, or charged, therapeuticagents. Exemplary ionic R^(y) moieties include polylysine, polyarginine,poly aspartic acid, polyhistidine, and polyglutamic acid.

Exemplary ionic, or charged, therapeutic agents include DNA plasmids,short interfering RNAs (siRNAs), micro RNAs (miRNAs), short hairpin RNAs(shRNAs), antisense RNAs, and other RNA-based therapeutics. Other ionic,or charged, therapeutic agents include oligopeptides, peptides,monoclonal antibodies, cytokines, and other protein therapeutics.

In other embodiments, the present invention provides a drug-loadedmicelle comprising a multiblock copolymer of formula II:

wherein:

n is 10-2500;

m is 1 to 1000;

m′ is 1 to 1000;

R^(x) is a natural or unnatural amino acid side-chain group that iscrosslinked;

R^(y) is a hydrophobic or ionic, natural or unnatural amino acidside-chain group;

R¹ is —Z(CH₂CH₂Y)_(p)(CH₂)_(t)R³, wherein:

-   -   Z is —O—, —S—, or —CH₂—;    -   each Y is independently —O— or —S—;    -   p is 0-10;    -   t is 0-10; and    -   R³ is —N₃, —CN, a mono-protected amine, a di-protected amine, a        protected aldehyde, a protected hydroxyl, a protected carboxylic        acid, a protected thiol, a 9-30-membered crown ether, or an        optionally substituted group selected from aliphatic, a 5-8        membered saturated, partially unsaturated, or aryl ring having        0-4 heteroatoms independently selected from nitrogen, oxygen, or        sulfur, an 8-10 membered saturated, partially unsaturated, or        aryl bicyclic ring having 0-5 heteroatoms independently selected        from nitrogen, oxygen, or sulfur, or a detectable moiety;

Q is a valence bond or a bivalent, saturated or unsaturated, straight orbranched C₁₋₁₂ alkylene chain, wherein 0-6 methylene units of Q areindependently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—,—C(O)—, —SO—, —SO₂—, —NHSO₂—, —SO₂NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or—NHC(O)O—, wherein:

-   -   -Cy- is an optionally substituted 5-8 membered bivalent,        saturated, partially unsaturated, or aryl ring having 0-4        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, or an optionally substituted 8-10 membered bivalent        saturated, partially unsaturated, or aryl bicyclic ring having        0-5 heteroatoms independently selected from nitrogen, oxygen, or        sulfur;

R^(2a) is a mono-protected amine, a di-protected amine, —N(R⁴)₂,—NR⁴C(O)R⁴, —NR⁴C(O)N(R⁴)₂, —NR⁴C(O)OR⁴, or —NR⁴SO₂R⁴; and

each R⁴ is independently an optionally substituted group selected fromhydrogen, aliphatic, a 5-8 membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, an 8-10-membered saturated, partially unsaturated, oraryl bicyclic ring having 0-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or a detectable moiety, or:

-   -   two R⁴ on the same nitrogen atom are taken together with said        nitrogen atom to form an optionally substituted 4-7 membered        saturated, partially unsaturated, or aryl ring having 1-4        heteroatoms independently selected from nitrogen, oxygen, or        sulfur.

Embodiments with respect to each of the R¹, R^(2a), Q, R^(x), R^(y), n,m, and m′ groups of formula II, are as described in various classes andsubclasses, both singly and in combination, herein.

In certain embodiments, R^(x) is a crosslinked amino acid side-chaingroup and R^(y) is a hydrophobic amino acid side-chain group. Suchhydrophilic, or crosslinkable, amino acid side-chain groups includetyrosine, serine, cysteine, threonine, aspartic acid (also known asaspartate, when charged), glutamic acid (also known as glutamate, whencharged), asparagine, and glutamine. Such hydrophobic amino acidside-chain groups include a suitably protected tyrosine side-chain, asuitably protected serine side-chain, a suitably protected threonineside-chain, phenylalanine, alanine, valine, leucine, tryptophan,proline, benzyl and alkyl glutamates, or benzyl and alkyl aspartates ormixtures thereof. Such ionic amino acid side chain groups includes alysine side-chain, arginine side-chain, or a suitably protected lysineor arginine side-chain, an aspartic acid side chain, glutamic acidside-chain, or a suitably protected aspartic acid or glutamic acidside-chain. One of ordinary skill in the art would recognize thatprotection of a polar or hydrophilic amino acid side-chain can renderthat amino acid nonpolar. For example, a suitably protected tyrosinehydroxyl group can render that tyrosine nonpolar and hydrophobic byvirtue of protecting the hydroxyl group. Suitable protecting groups forthe hydroxyl, amino, and thiol, and carboylate functional groups ofR^(x) and R^(y) are as described herein.

In other embodiments, R^(y) comprises a mixture of hydrophobic andhydrophilic amino acid side-chain groups such that the overallpoly(amino acid) block comprising R^(y) is hydrophobic. Such mixtures ofamino acid side-chain groups include phenylalanine/tyrosine,phenalanine/serine, leucine/tyrosine, and the like. According to anotherembodiment, R^(y) is a hydrophobic amino acid side-chain group selectedfrom phenylalanine, alanine, or leucine, and one or more of tyrosine,serine, or threonine.

As defined above, R^(x) is a crosslinked natural or unnatural amino acidside-chain group. It will be appreciated that a variety of amino acidside-chain functional groups are capable of such cross-linking,including, but not limited to, carboxylate, hydroxyl, thiol, and aminogroups. Examples of R^(x) moieties having functional groups capable offorming cross-links include a glutamic acid side-chain, —CH₂C(O)CH, anaspartic acid side-chain, —CH₂CH₂C(O)OH, a cystein side-chain, —CH₂SH, aserine side-chain, —CH₂OH, an aldehyde containing side-chain, —CH₂C(O)H,a lysine side-chain, —(CH₂)₄NH₂, an arginine side-chain,—(CH₂)₃NHC(═NH)NH₂, a histidine side-chain, —CH₂-imidazol-4-yl.

In still other embodiments, the present invention provides a drug-loadedmicelle comprising a multiblock copolymer of formula III:

wherein:

n is 10-2500;

m is 1 to 1000;

m′ is 1 to 1000;

L is a bivalent, saturated or unsaturated, straight or branched C₁₋₁₂alkylene chain, wherein 0-6 methylene units of L are independentlyreplaced by -M-, -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—,—SO₂—, —NHSO₂—, —SO₂NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or —NHC(O)O—,wherein:

-   -   -M- is a suitable bivalent metal;    -   -Cy- is an optionally substituted 5-8 membered bivalent,        saturated, partially unsaturated, or aryl ring having 0-4        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, or an optionally substituted 8-10 membered bivalent        saturated, partially unsaturated, or aryl bicyclic ring having        0-5 heteroatoms independently selected from nitrogen, oxygen, or        sulfur;

R^(y) is a hydrophobic or ionic, natural or unnatural amino acidside-chain group;

R¹ is —Z(CH₂CH₂Y)_(p)(CH₂)_(t)R³, wherein:

-   -   -   Z is —O—, —S—, —C≡C—, or —CH₂—;

    -   each Y is independently —O— or —S—;

    -   p is 0-10;

    -   t is 0-10; and

    -   R³ is —N₃, —CN, a mono-protected amine, a di-protected amine, a        protected aldehyde, a protected hydroxyl, a protected carboxylic        acid, a protected thiol, a 9-30 membered crown ether, or an        optionally substituted group selected from aliphatic, a 5-8        membered saturated, partially unsaturated, or aryl ring having        0-4 heteroatoms independently selected from nitrogen, oxygen, or        sulfur, an 8-10 membered saturated, partially unsaturated, or        aryl bicyclic ring having 0-5 heteroatoms independently selected        from nitrogen, oxygen, or sulfur, or a detectable moiety;

Q is a valence bond or a bivalent, saturated or unsaturated, straight orbranched C₁₋₁₂ alkylene chain, wherein 0-6 methylene units of Q areindependently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—,—C(O)—, —SO—, —SO₂—, —NHSO₂—, —SO₂NH—, —NHC(O)—, —C(O)NH—,—OC(O)NH—, or—NHC(O)O—, wherein:

-   -   -Cy- is an optionally substituted 5-8 membered bivalent,        saturated, partially unsaturated, or aryl ring having 0-4        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, or an optionally substituted 8-10 membered bivalent        saturated, partially unsaturated, or aryl bicyclic ring having        0-5 heteroatoms independently selected from nitrogen, oxygen, or        sulfur;

R^(2a) is a mono-protected amine, a di-protected amine, —N(R⁴)₂,—NR⁴C(O)R⁴, —NR⁴C(O)N(R⁴)₂, —NR⁴C(O)OR⁴, or —NR⁴SO₂R⁴; and

each R⁴ is independently an optionally substituted group selected fromhydrogen, aliphatic, a 5-8 membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, oraryl bicyclic ring having 0-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or a detectable moiety, or:

-   -   two R⁴ on the same nitrogen atom are taken together with said        nitrogen atom to form an optionally substituted 4-7 membered        saturated, partially unsaturated, or aryl ring having 1-4        heteroatoms independently selected from nitrogen, oxygen, or        sulfur.

Embodiments with respect to each of the R¹, R^(2a), L, Q, R^(y), n, m,and m′ groups of formula III, are as described in various classes andsubclasses, both singly and in combination, herein.

In certain embodiments, R^(y) is a hydrophobic amino acid side-chaingroup. Such hydrophobic amino acid side-chain groups include a suitablyprotected tyrosine side-chain, a suitably protected serine side-chain, asuitably protected threonine side-chain, phenylalanine, alanine, valine,leucine, tryptophan, proline, benzyl and alkyl glutamates, or benzyl andalkyl aspartates or mixtures thereof. Such ionic amino acid side chaingroups includes a lysine side-chain, arginine side-chain, or a suitablyprotected lysine or arginine side-chain, an aspartic acid side chain,glutamic acid side-chain, or a suitably protected aspartic acid orglutamic acid side-chain. One of ordinary skill in the art wouldrecognize that protection of a polar or hydrophilic amino acidside-chain can render that amino acid nonpolar. For example, a suitablyprotected tyrosine hydroxyl group can render that tyrosine nonpolar andhydrophobic by virtue of protecting the hydroxyl group. Suitableprotecting groups for the hydroxyl, amino, and thiol functional groupsof R^(y) are as described herein.

In other embodiments, R^(y) comprises a mixture of hydrophobic andhydrophilic amino acid side-chain groups such that the overallpoly(amino acid) block comprising R^(y) is hydrophobic. Such mixtures ofamino acid side-chain groups include phenylalanine/tyrosine,phenalanine/serine, leucine/tyrosine, and the like. According to anotherembodiment, R^(y) is a hydrophobic amino acid side-chain group selectedfrom phenylalanine, alanine, or leucine, and one or more of tyrosine,serine, or threonine.

In certain embodiments, micelles of the present invention are loadedwith a hydrophobic drug. In accordance with such embodiments, the R^(y)group of formula III is a natural or unnatural hydrophobic amino acidside-chain group. Such hydrophobic amino acid side-chain groups includea suitably protected tyrosine side-chain, a suitably protected serineside-chain, a suitably protected threonine side-chain, phenylalanine,alanine, valine, leucine, tryptophan, proline, benzyl and alkylglutamates, or benzyl and alkyl aspartates, or mixtures thereof. One ofordinary skill in the art would recognize that protection of a polar orhydrophilic amino acid side-chain can render that amino acid nonpolar.For example, a suitably protected tyrosine hydroxyl group can renderthat tyrosine nonpolar and hydrophobic by virtue of protecting thehydroxyl group. Suitable protecting groups for the hydroxyl, amino, andthiol, and carboxylate functional groups of R^(y) are as describedherein.

In certain embodiments, the R^(y) group of formula III comprises amixture of hydrophobic and hydrophilic amino acid side-chain groups suchthat the overall poly(amino acid) block comprising R^(y) is hydrophobic.In other embodiments, R^(y) comprises a mixture of phenylalanine andtyrosine. By way of example, this particular copolymer is used toencapsulate one or more of DOX, CPT, and paclitaxel in the hydrophobicphenylalanine/tyrosine inner core. Although only sparingly soluble inwater, these drugs possess polar functionalities (e.g. amine, alcohol,and phenols), which makes the incorporation of tyrosine, a polar aminoacid, advantageous for effective encapsulation. By utilizing thisparticular core composition, relatively high DOX, CPT, and paclitaxelloadings are achieved. In certain embodiments, the present inventionprovides a micelle comprising a compound of formula III characterized inthat DOX, CPT, and paclitaxel are encapsulated in the hydrophobicphenylalanine/tyrosine inner core and the poly(aspartic acid) outer coreis crosslinked with zinc. In certain embodiments, m and m′ add up toabout 30 to about 60. In still other embodiments, m is 1-20 repeat unitsand m′ is 10-50 repeat units. In certain embodiments, thephenylalanine/tyrosine ratio of m′ is 4:1. In other embodiments the thephenylalanine/tyrosine ratio of m′ is 9:1. In still other embodiments,the phenylalanine/tyrosine ratio of m′ is 3:1. In other embodiments,R^(y) comprises 4-8 tyrosine repeat units and 20-32 phenylalanine. Instill other embodiments, R^(y) comprises 2-40 tyrosine and 10-100phenylalanine repeat units.

Hydrophobic small molecule drugs suitable for loading into micelles ofthe present invention are well known in the art. In certain embodiments,the present invention provides a drug-loaded micelle as describedherein, wherein the drug is a hydrophobic drug selected from analgesics,anti-inflammatory agents, antihelminthics, anti-arrhythmic agents,anti-bacterial agents, anti-viral agents, anti-coagulants,anti-depressants, anti-diabetics, anti-epileptics, anti-fungal agents,anti-gout agents, anti-hypertensive agents, anti-malarials,anti-migraine agents, anti-muscarinic agents, anti-neoplastic agents,erectile dysfunction improvement agents, immunosuppressants,anti-protozoal agents, anti-thyroid agents, anxiolytic agents,sedatives, hypnotics, neuroleptics, β-blockers, cardiac inotropicagents, corticosteroids, diuretics, anti-parkinsonian agents,gastro-intestinal agents, histamine receptor antagonists, keratolyptics,lipid regulating agents, anti-anginal agents, Cox-2 inhibitors,leukotriene inhibitors, macrolides, muscle relaxants, nutritionalagents, opiod analgesics, protease inhibitors, sex hormones, stimulants,muscle relaxants, anti-osteoporosis agents, anti-obesity agents,cognition enhancers, anti-urinary incontinence agents, anti-benignprostate hypertrophy agents, essential fatty acids, non-essential fattyacids, and mixtures thereof.

In other embodiments, the hydrophobic drug is selected from one or moreanalgesics, anti-bacterial agents, anti-viral agents, anti-inflammatoryagents, anti-depressants, anti-diabetics, anti-epileptics,anti-hypertensive agents, anti-migraine agents, immunosuppressants,anxiolytic agents, sedatives, hypnotics, neuroleptics, 13-blockers,gastro-intestinal agents, lipid regulating agents, anti-anginal agents,Cox-2 inhibitors, leukotriene inhibitors, macrolides, muscle relaxants,opioid analgesics, protease inhibitors, sex hormones, cognitionenhancers, anti-urinary incontinence agents, and mixtures thereof.

According to one aspect, the present invention provides a micelle, asdescribed herein, loaded with a hydrophobic drug selected from any oneor more of acetretin, albendazole, albuterol, aminoglutethimide,amiodarone, amlodipine, amphetamine, amphotericin B, atorvastatin,atovaquone, azithromycin, baclofen, beclomethasone, benezepril,benzonatate, betamethasone, bicalutanide, budesonide, bupropion,busulfan, butenafine, calcifediol, calcipotriene, calcitriol,camptothecin, candesartan, capsaicin, carbamezepine, carotenes,celecoxib, cerivastatin, cetirizine, chlorpheniramine, cholecalciferol,cilostazol, cimetidine, cinnarizine, ciprofloxacin, cisapride,clarithromycin, clemastine, clomiphene, clomipramine, clopidogrel,codeine, coenzyme Q10, cyclobenzaprine, cyclosporin, danazol,dantrolene, dexchlorpheniramine, diclofenac, dicoumarol, digoxin,dehydroepiandrosterone, dihydroergotamine, dihydrotachysterol,dirithromycin, donezepil, efavirenz, eprosartan, ergocalciferol,ergotamine, essential fatty acid sources, etodolac, etoposide,famotidine, fenofibrate, fentanyl, fexofenadine, finasteride,fluconazole, flurbiprofen, fluvastatin, fosphenytoin, frovatriptan,furazolidone, gabapentin, gemfibrozil, glibenclamide, glipizide,glyburide, glimepiride, griseofulvin, halofantrine, ibuprofen,irbesartan, irinotecan, isosorbide dinitrate, isotretinoin,itraconazole, ivermectin, ketoconazole, ketorolac, lamotrigine,lansoprazole, leflunomide, lisinopril, loperamide, loratadine,lovastatin, L-thryroxine, lutein, lycopene, medroxyprogesterone,mifepristone, mefloquine, megestrol acetate, methadone, methoxsalen,metronidazole, miconazole, midazolam, miglitol, minoxidil, mitoxantrone,montelukast, nabumetone, nalbuphine, naratriptan, nelfinavir,nifedipine, nilsolidipine, nilutanide, nitrofurantoin, nizatidine,omeprazole, oprevelkin, oestradiol, oxaprozin, paclitaxel, paracalcitol,paroxetine, pentazocine, pioglitazone, pizofetin, pravastatin,prednisolone, probucol, progesterone, pseudoephedrine, pyridostigmine,rabeprazole, raloxifene, rofecoxib, repaglinide, rifabutine,rifapentine, rimexolone, ritanovir, rizatriptan, rosiglitazone,saquinavir, sertraline, sibutramine, sildenafil citrate, simvastatin,sirolimus, spironolactone, sumatriptan, tacrine, tacrolimus, tamoxifen,tamsulosin, targretin, tazarotene, telmisartan, teniposide, terbinafine,terazosin, tetrahydrocannabinol, tiagabine, ticlopidine, tirofibran,tizanidine, topiramate, topotecan, toremitfene, tramadol, tretinoin,troglitazone, trovafloxacin, ubidecarenone, valsartan, venlafaxine,verteporfin, vigabatrin, vitamin A, vitamin D, vitamin E, vitamin K,zafirlukast, zileuton, zolmitriptan, zolpidem, zopiclone,pharmaceutically acceptable salts, isomers, and derivatives thereof, andmixtures thereof.

According to another embodiment, the present invention provides amicelle, as described herein, loaded with a hydrophobicantiproliferative or chemotherapeutic drug. One of ordinary skill in theart will appreciate that many anticancer agents are hydrophobic. Incertain embodiments, the hydrophobic antiproliferative orchemotherapeutic drug is selected from any one or more of a taxane(e.g., paclitaxel), vincristine, adriamycin, vinca alkaloids (e.g.,vinblastine), anthracyclines (e.g., doxorubicin), epipodophyllotoxins(e.g., etoposide), cisplatin, methotrexate, actinomycin D, actinomycinD, dolastatin 10, colchicine, emetine, trimetrexate, metoprine,cyclosporine, daunorubicin, teniposide, amphotericin, alkylating agents(e.g., chlorambucil), 5-fluorouracil, campthothecin, cisplatin, andmetronidazole, among others.

In certain embodiments, the present invention provides a micelle, asdescribed herein, loaded with an antiproliferative or chemotherapeuticagent selected from any one or more of Abarelix, aldesleukin,Aldesleukin, Alemtuzumab, Alitretinoin, Allopurinol, Altretamine,Amifostine, Anastrozole, Arsenic trioxide, Asparaginase, Azacitidine,BCG Live, Bevacuzimab, Avastin, Fluorouracil, Bexarotene, Bleomycin,Bortezomib, Busulfan, Calusterone, Capecitabine, Camptothecin,Carboplatin, Carmustine, Celecoxib, Cetuximab, Chlorambucil, Cisplatin,Cladribine, Clofarabine, Cyclophosphamide, Cytarabine, Dactinomycin,Darbepoetin alfa, Daunorubicin, Denileukin, Dexrazoxane, Docetaxel,Doxorubicin (neutral), Doxorubicin hydrochloride, DromostanolonePropionate, Epirubicin, Epoetin alfa, Erlotinib, Estramustine, EtoposidePhosphate, Etoposide, Exemestane, Filgrastim, floxuridine fludarabine,Fulvestrant, Gefitinib, Gemcitabine, Gemtuzumab, Goserelin Acetate,Histrelin Acetate, Hydroxyurea, Ibritumomab, Idarubicin, Ifosfamide,Imatinib Mesylate, Interferon Alfa-2a, Interferon Alfa-2b, Irinotecan,Lenalidomide, Letrozole, Leucovorin, Leuprolide Acetate, Levamisole,Lomustine, Megestrol Acetate, Melphalan, Mercaptopurine, 6-MP, Mesna,Methotrexate, Methoxsalen, Mitomycin C, Mitotane, Mitoxantrone,Nandrolone, Nelarabine, Nofetumomab, Oprelvekin, Oxaliplatin,Paclitaxel, Palifermin, Pamidronate, Pegademase, Pegaspargase,Pegfilgrastim, Pemetrexed Disodium, Pentostatin, Pipobroman, Plicamycin,Porfimer Sodium, Procarbazine, Quinacrine, Rasburicase, Rituximab,Sargramostim, Sorafenib, Streptozocin, Sunitinib Maleate, Talc,Tamoxifen, Temozolomide, Teniposide, VM-26, Testolactone, Thioguanine,6-TG, Thiotepa, Topotecan, Toremifene, Tositumomab, Trastuzumab,Tretinoin, ATRA, Uracil Mustard, Valrubicin, Vinblastine, Vincristine,Vinorelbine, Zoledronate, or Zoledronic acid.

According to another embodiment, the present invention provides amicelle, as described herein, loaded with a treatment for Alzheimer'sDisease such as Aricept® or Excelon®; a treatment for Parkinson'sDisease such as L-DOPA/carbidopa, entacapone, ropinrole, pramipexole,bromocriptine, pergolide, trihexephendyl, or amantadine; an agent fortreating Multiple Sclerosis (MS) such as beta interferon (e.g., Avonex®and Rebif®), Copaxone®, or mitoxantrone; a treatment for asthma such asa steroid, albuterol or Singulair®; an agent for treating schizophreniasuch as zyprexa, risperdal, seroquel, or haloperidol; ananti-inflammatory agent such as corticosteroids, TNF blockers, IL-1 RA,azathioprine, cyclophosphamide, or sulfasalazine; an immunomodulatoryand immunosuppressive agent such as cyclosporin, tacrolimus, rapamycin,mycophenolate mofetil, interferons, corticosteroids, cyclophophamide,azathioprine, or sulfasalazine; a neurotrophic factor such asacetylcholinesterase inhibitors, MAO inhibitors, interferons,anti-convulsants, ion channel blockers, riluzole, or anti-Parkinsonianagents; an agent for treating cardiovascular disease such asbeta-blockers, ACE inhibitors, diuretics, nitrates, calcium channelblockers, or statins; an agent for treating liver disease such ascorticosteroids, cholestyramine, interferons, or anti-viral agents; anagent for treating blood disorders such as corticosteroids,anti-leukemic agents, or growth factors; and an agent for treatingimmunodeficiency disorders such as gamma globulin.

In other embodiments, when the R^(y) group of formula III is an ionicnatural or unnatural amino acid side-chain, micelles of the presentinvention are useful for encapsulating ionic, or charged, therapeuticagents. Exemplary ionic R^(y) moieties include polylysine, polyarginine,poly aspartic acid, polyhistidine, and polyglutamic acid.

Exemplary ionic, or charged, therapeutic agents include DNA plasmids,short interfering RNAs (siRNAs), micro RNAs (miRNAs), short hairpin RNAs(shRNAs), antisense RNAs, and other RNA-based therapeutics. Other ionic,or charged, therapeutic agents include oligopeptides, peptides,monoclonal antibodies, cytokines, and other protein therapeutics.

Targeting the delivery of potent, cytotoxic agents specifically tocancer cells using responsive nanovectors would have a clear impact onthe well-being of the many thousands of people who rely on traditionalsmall molecule therapeutics for the treatment of cancer. In certainembodiments, the present invention provides micelle-encapsulated formsof the common chemotherapy drugs, doxorubicin (adriamycin), atopoisomerase II inhibitor, camptothecin (CPT), a topoisomerase Iinhibitor, or paclitaxel (Taxol), an inhibitor of microtubule assembly.These drugs are both effective chemotherapy agents but suffer fromclinical problems which are effectively addressed by cancer-specificdelivery. For example, the cytotoxic nature of these drugs affectstumors and healthy tissue alike, resulting in a multitude of sideeffects such as dermatitis, hair loss, and nausea. DOX side effects suchas acute cardiotoxicity and bone marrow suppression are particularlyproblematic. Neutral doxorubicin, camptothecin, and paclitaxel arepoorly soluble in water (i.e., hydrophobic), making them candidates formicellar delivery. Camptothecin, which possesses a hydrolyticallydegradable lactone ring, has a short half-life in aqueous solution,especially at elevated pH. Without wishing to be bound by any particulartheory, it is believed that encapsulation in the hydrophobic micellecore will significantly increase the half-life of the drug. A multitudeof drug delivery systems have been employed to reduce the aforementionedproblems associated with doxorubicin, camptothecin, and paclitaxel, withvarying degrees of success.

D. Polymer Conjugation

In addition to their core-shell morphology, polymer micelles can bemodified to enable passive and active cell-targeting to maximize thebenefits of current and future therapeutic agents. Because drug-loadedmicelles typically possess diameters greater than 20 nm, they exhibitdramatically increased circulation time when compared to stand-alonedrugs due to minimized renal clearance. This unique feature ofnanovectors and polymeric drugs leads to selective accumulation indiseased tissue, especially cancerous tissue due to the enhancedpermeation and retention effect (“EPR”). The EPR effect is a consequenceof the disorganized nature of the tumor vasculature, which results inincreased permeability of polymer therapeutics and drug retention at thetumor site. In addition to passive cell targeting by the EPR effect,micelles are designed to actively target tumor cells through thechemical attachment of targeting groups to the micelle periphery. Theincorporation of such groups is most often accomplished throughend-group functionalization of the hydrophilic block using chemicalconjugation techniques. Like viral particles, micelles functionalizedwith targeting groups utilize receptor-ligand interactions to controlthe spatial distribution of the micelles after administration, furtherenhancing cell-specific delivery of therapeutics. In cancer therapy,targeting groups are designed to interact with receptors that areover-expressed in cancerous tissue relative to normal tissue such asfolic acid, oligopeptides, sugars, and monoclonal antibodies. See Pan,D.; Turner, J. L.; Wooley, K. L. Chem. Commun. 2003, 2400-2401; Gabizon,A.; Shmeeda, H.; Horowitz, A. T.; Zalipsky, S. Adv. Drug Deliv. Rev.2004, 56, 1177-1202; Reynolds, P. N.; Dmitriev, I.; Curiel, D. T.Vector. Gene Ther. 1999, 6, 1336-1339; Derycke, A. S. L.; Kamuhabwa, A.;Gijsens, A.; Roskams, T.; De Vos, D.; Kasran, A.; Huwyler, J.; Missiaen,L.; de Witte, P. A. M. T J. Nat. Cancer Inst. 2004, 96, 1620-30;Nasongkla, N., Shuai, X., Ai, H.,; Weinberg, B. D. P., J.; Boothman, D.A.; Gao, J. Angew. Chem. Int. Ed. 2004, 43, 6323-6327; Jule, E.;Nagasaki, Y.; Kataoka, K. Bioconj. Chem. 2003, 14, 177-186; Stubenrauch,K.; Gleiter, S.; Brinkmann, U.; Rudolph, R.; Lilie, H. Biochem. J. 2001,356, 867-873; Kurschus, F. C.; Kleinschmidt, M.; Fellows, E.; Dornmair,K.; Rudolph, R.; Lilie, H.; Jenne, D. E. FEBS Lett. 2004, 562, 87-92;and Jones, S. D.; Marasco, W. A. Adv. Drug Del. Rev. 1998, 31, 153-170.

Compounds of any of formulae I, II, and III having R³ moieties suitablefor Click chemistry are useful for conjugating said compounds tobiological systems or macromolecules such as proteins, viruses, andcells, to name but a few. The Click reaction is known to proceed quicklyand selectively under physiological conditions. In contrast, mostconjugation reactions are carried out using the primary aminefunctionality on proteins (e.g. lysine or protein end-group). Becausemost proteins contain a multitude of lysines and arginines, suchconjugation occurs uncontrollably at multiple sites on the protein. Thisis particularly problematic when lysines or arginines are located aroundthe active site of an enzyme or other biomolecule. Thus, anotherembodiment of the present invention provides a method of conjugating theR¹ groups of a compound of any of formulae I, II, and III to amacromolecule via Click chemistry. Yet another embodiment of the presentinvention provides a macromolecule conjugated to a compound of any offormulae I, II, and III via the R¹ group.

After incorporating the poly (amino acid) block portions into themulti-block coploymer of the present invention resulting in amulti-block copolymer of the form W—X—X′, the other end-groupfunctionality, corresponding to the R¹ moiety of any of formulae I, II,and III, can be used to attach targeting groups for cell specificdelivery including, but not limited to, attach targeting groups for cellspecific delivery including, but not limited to, proteins,oliogopeptides, antibodies, monosaccarides, oligosaccharides, vitamins,or other small biomolecules. Such targeting groups include, but or notlimited to monoclonal and polyclonal antibodies (e.g. IgG, IgA, IgM,IgD, IgE antibodies), sugars (e.g. mannose, mannose-6-phosphate,galactose), proteins (e.g. Transferrin), oligopeptides (e.g. cyclic andacylic RGD-containing oligopedptides), and vitamins (e.g. folate).Alternatively, the R¹ moiety of any of formulae I, II, and III is bondedto a biomolecule, drug, cell, or other suitable substrate.

In other embodiments, the R¹ moiety of any of formulae I, II, and III isbonded to biomolecules which promote cell entry and/or endosomal escape.Such biomolecules include, but are not limited to, oligopeptidescontaining protein transduction domains such as the HIV Tat peptidesequence (GRKKRRQRRR) or oligoarginine (RRRRRRRRR). Oligopeptides whichundergo conformational changes in varying pH environments sucholigohistidine (HHHHH) also promote cell entry and endosomal escape.

In other embodiments, the R¹ moiety of any of formulae I, II, and III isbonded to detectable moieties, such as fluorescent dyes or labels forpositron emission tomography including molecules containingradioisotopes (e.g. ¹⁸F) or ligands with bound radioactive metals (e.g.⁶²Cu).) In other embodiments, the R¹ moiety of any of formulae I, II,and III is bonded to a contrast agents for magnetic resonance imagingsuch as gadolinium, gadolinium chelates, or iron oxide (e.g Fe₃O₄ andFe₂O₃) particles. In other embodiments, the R¹ moiety of any of formulaeI, II, and III is bonded to a semiconducting nanoparticle such ascadmium selenide, cadmium sulfide, or cadmium telluride or bonded toother metal nanoparticles such as colloidal gold. In other embodiments,the R¹ moiety of any of formulae I, II, and III is bonded to natural orsynthetic surfaces, cells, viruses, dyes, drugs, chelating agents, orused for incorporation into hydrogels or other tissue scaffolds.

In one embodiment, the R¹ moiety of any of formulae I, II, and III is anacetylene or an acetylene derivative which is capable of undergoing[3+2] cycloaddition reactions with complementary azide-bearing moleculesand biomolecules. In another embodiment, the R¹ moiety of any offormulae I, II, and III is an azide or an azide derivative which iscapable of undergoing [3+2] cycloaddition reactions with complementaryalkyne-bearing molecules and biomolecules (i.e. click chemistry).

Click chemistry has become a popular method of bioconjugation due to itshigh reactivity and selectivity, even in biological media. See Kolb, H.C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40,2004-2021; and Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.;Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192-3193. Inaddition, currently available recombinant techniques permit theintroduction of azides and alkyne-bearing non-canonical amino acids intoproteins, cells, viruses, bacteria, and other biological entities thatconsist of or display proteins. See Link, A. J.; Vink, M. K. S.;Tirrell, D. A. J. Am. Chem. Soc. 2004, 126, 10598-10602; Deiters, A.;Cropp, T. A.; Mukherji, M.; Chin, J. W.; Anderson, C.; Schultz, P. G. J.Am. Chem. Soc. 2003, 125, 11782-11783.

In another embodiment, the [3+2] cycloaddition reaction of azide oracetylene-bearing nanovectors and complimentary azide oracetylene-bearing biomolecules are transition metal catalyzed.Copper-containing molecules which catalyze the “click” reaction include,but are not limited to, copper bromide (CuBr), copper chloride (CuCl),copper sulfate (CuSO₄), copper iodide (CuI), [Cu(MeCN)₄](OTO, and[Cu(MeCN)₄](PF₆). Organic and inorganic metal-binding ligands can beused in conjunction with metal catalysts and include, but are notlimited to, sodium ascorbate, tris(triazolyl)amine ligands,tris(carboxyethyl)phosphine (TCEP), and sulfonated bathophenanthrolineligands.

In another embodiment, the R¹ moiety of any of formulae I, II, and IIIis an hydrazine or hydrazide derivative which is capable of undergoingreaction with biomolecules containing aldehydes or ketones to formhydrazone linkages. In another embodiment, the R¹ moiety of any offormulae I, II, and III is an aldehyde or ketone derivative which iscapable of undergoing reaction with biomolecules containing a hydrazineor hydrazide derivative to form hydrazone linkages.

In another embodiment, the R¹ moiety of any of formulae I, II, and IIIis a hydroxylamine derivative which is capable of undergoing reactionwith biomolecules containing aldehydes or ketones. In anotherembodiment, the R¹ moiety of any of formulae I, II, and III is analdehyde or ketone which is capable of undergoing reaction withbiomolecules containing a hydroxylamine, or a hydroxylamine derivative.

In yet another embodiment, the R¹ moiety of any of formulae I, II, andIII is an aldehyde or ketone derivative which is capable of undergoingreaction with biomolecules containing primary or secondary amines toform imine linkages. In another embodiment, the R¹ moiety of any offormulae I, II, and III is a primary or secondary amine which is capableof undergoing reaction with biomolecules containing an aldehyde orketone functionality to form imine linkages. It will be appreciated thatimine linkages can be further converted to stable amine linkages bytreatment with a suitable reducing agent (e.g. lithium aluminum hydride,sodium borohydride, sodium cyanoborohydride, etc.)

In yet another embodiment, the R¹ moiety of any of formulae I, II, andIII is an amine (primary or secondary) or alcohol which is capable ofundergoing reaction with biomolecules containing activated esters (e.g.4-nitrophenol ester, N-hydroxysuccinimide, pentafluorophenyl ester,ortho-pyridylthioester), to form amide or ester linkages. In still otherembodiments, the R¹ moiety of any of formulae I, II, and III is anactivated ester which is capable of undergoing reaction withbiomolecules possessing amine (primary or secondary) or alcohols to formamide or ester linkages.

In still other embodiments, the R¹ moiety of any of formulae I, II, andIII is an amine or alcohol which is bound to biomolecules withcarboxylic acid functionality using a suitable coupling agent. In stillother embodiments, the R¹ moiety of any of formulae I, II, and III is acarboxylic acid functionality which is bound to biomolecules containingamine or alcohol functionality using a suitable coupling agent. Suchcoupling agents include, but are not limited to, carbodiimides (e.g.1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), diisopropylcarbodiimide (DIC), dicyclohexyl carbodiimide (DCC)), aminium orphosphonium derivatives (e.g. PyBOP, PyAOP, TBTU, HATU, HBTU), or acombination of 1-hydroxybenzotriazole (HOBt) and a aminium orphosphonium derivative.

In another embodiment, the R¹ moiety of any of formulae I, II, and IIIis an electrophile such as maleimide, a maleimide derivative, or abromoacetamide derivative, which is capable of reaction withbiomolecules containing thiols or amines. In another embodiment, the R¹moiety of any of formulae I, II, and III is a nucleophile such as anamine or thiol which is capable or reaction with biomolecules containingelectrophilic functionality such as maleimide, a maleimide derivative,or a bromoacetamide derivative.

In still other embodiments, the R¹ moiety of any of formulae I, II, andIII is a ortho-pyridyl disulfide moiety which undergoes disulfideexchange with biomolecules containing thiol functionality. In stillother embodiments, the R¹ moiety of any of formulae I, II, and III is athiol or thiol derivative which undergoes disulfide exchange withbiomolecules containing ortho-pyridyl disulfide functionality. It willbe appreciated that such exchange reactions result in a disulfidelinkage which is reversible in the presence of a suitable reducing agent(e.g. glutathione, dithiothreitol (DTT), etc.).

In certain embodiments, micelles of the present invention are mixedmicelles comprising one or more compounds of formula I, II, or III. Itwill be appreciated that mixed micelles having different R¹ groups, asdescribed herein, can be conjugated to multiple other compounds and/ormacromolecules. For example, a mixed micelle of the present inventioncan have one R¹ group suitable for Click chemistry and another R¹ groupsuitable for covalent attachment via a variety of coupling reactions.Such a mixed micelle can be conjugated to different compounds and/ormacromolecules via these different R¹ groups. Such conjugation reactionsare well known to one of ordinary skill in the art and include thosedescribed herein.

4. General Methods for Providing Compounds of the Present Invention

Multiblock copolymers of the present invention are prepared by methodsknown to one of ordinary skill in the art and those described in detailin U.S. patent application Ser. No. 11/325,020 filed Jan. 4, 2006, theentirety of which is hereby incorporated herein by reference. Generally,such multiblock copolymers are prepared by sequentially polymerizing oneor more cyclic amino acid monomers onto a hydrophilic polymer having aterminal amine salt wherein said polymerization is initiated by saidamine salt. In certain embodiments, said polymerization occurs byring-opening polymerization of the cyclic amino acid monomers. In otherembodiments, the cyclic amino acid monomer is an amino acid NCA, lactam,or imide.

Scheme 2 above depicts a general method for preparing multiblockpolymers of the present invention. A macroinitiator of formula A istreated with a first amino acid NCA to form a compound of formula Bhaving a first amino acid block. The second amino acid NCA is added tothe living polymer of formula B to form a compound of formula I′ havingtwo differing amino acid blocks. Each of the R¹, A, n, Q, R^(x), R^(y),m, and m′ groups depicted in Scheme 2 are as defined and described inclasses and subclasses, singly and in combination, herein.

One step in the preparation of a compound of formula I comprisesterminating the living polymer chain-end of the compound of formula I′with a suitable polymerization terminator to afford a compound offormula I. One of ordinary skill in the art would recognize that thepolymerization terminator provides the R^(2a) group of formula I.Accordingly, embodiments directed to the R^(2a) group of formula I asset forth above and herein, are also directed to the suitablepolymerization terminator itself, and similarly, embodiments directed tothe suitable polymerization terminator, as set forth above and herein,are also directed to the R^(2a) group of formula I.

As described above, compounds of formula I are prepared from compoundsof formula I′ by treatment with a suitable terminating agent. One ofordinary skill in the art would recognize that compounds of formula Iare also readily prepared directly from compounds of formula I′. In suchcases, and in certain embodiments, the compound of formula I′ is treatedwith a base to form the freebase compound prior to, or concurrent with,treatment with the suitable terminating agent. For example, it iscontemplated that a compound of formula I′ is treated with a base andsuitable terminating agent in the same reaction to form a freebase ofthat compound. In such cases, it is also contemplated that the base mayalso serve as the reaction medium.

One of ordinary skill in the art would also recognize that the abovemethod for preparing a compound of formula I may be performed as a“one-pot” synthesis of compounds of formula I that utilizes the livingpolymer chain-end to incorporate the R² group of formula I.Alternatively, compounds of formula I may also be prepared in amulti-step fashion. For example, the living polymer chain-end of acompound of formula I′ may be quenched to afford an amino group whichmay then be further derivatized, according to known methods, to afford acompound of formula I.

One of ordinary skill in the art will recognize that a variety ofpolymerization terminating agents are suitable for the presentinvention. Such polymerization terminating agents include anyR^(2a)-containing group capable of reacting with the living polymerchain-end of a compound of formula I′, or the free-based amino group offormula I′, to afford a compound of formula I. Thus, polymerizationterminating agents include anhydrides, and other acylating agents, andgroups that contain a suitable leaving group LG that is subject tonucleophilic displacement.

Alternatively, compounds of formula I′ may be coupled to carboxylicacid-containing groups to form an amide thereof. Thus, it iscontemplated that the amine group of formula I′ or freease thereof, maybe coupled with a carboxylic acid moiety to afford compounds of formulaI wherein R^(2a) is —NHC(O)R⁴. Such coupling reactions are well known inthe art. In certain embodiments, the coupling is achieved with asuitable coupling reagent. Such reagents are well known in the art andinclude, for example, DCC and EDC, among others. In other embodiments,the carboxylic acid moiety is activated for use in the couplingreaction. Such activation includes formation of an acyl halide, use of aMukaiyama reagent, and the like. These methods, and others, are known toone of ordinary skill in the art, e.g., see, “Advanced OrganicChemistry,” Jerry March, 5^(th) Ed., pp. 351-357, John Wiley and Sons,N.Y.

A “suitable leaving group that is subject to nucleophilic displacement”is a chemical group that is readily displaced by a desired incomingchemical moiety. Suitable leaving groups are well known in the art,e.g., see, March. Such leaving groups include, but are not limited to,halogen, alkoxy, sulphonyloxy, optionally substituted alkylsulphonyloxy,optionally substituted alkenylsulfonyloxy, optionally substitutedarylsulfonyloxy, and diazonium moieties. Examples of suitable leavinggroups include chloro, iodo, bromo, fluoro, methanesulfonyloxy(mesyloxy), tosyloxy, triflyloxy, nitro-phenylsulfonyloxy (nosyloxy),and bromo-phenylsulfonyloxy (brosyloxy).

According to an alternate embodiment, the suitable leaving group may begenerated in situ within the reaction medium. For example, a leavinggroup may be generated in situ from a precursor of that compound whereinsaid precursor contains a group readily replaced by said leaving groupin situ.

Alternatively, when the R^(2a) group of formula I is a mono- or di-protected amine, the protecting group(s) is removed and that functionalgroup may be derivatized or protected with a different protecting group.It will be appreciated that the removal of any protecting group of theR^(2a) group of formula I is performed by methods suitable for thatprotecting group. Such methods are described in detail in Green.

In other embodiments, the R^(2a) group of formula I is incorporated byderivatization of the amino group of formula I′, or freebase thereof,via anhydride coupling, optionally in the presence of base asappropriate. One of ordinary skill in the art would recognize thatanhydride polymerization terminating agents containing an azide, analdehyde, a hydroxyl, an alkyne, and other groups, or protected formsthereof, may be used to incorporate said azide, said aldehyde, saidprotected hydroxyl, said alkyne, and other groups into the R^(2a) groupof compounds of formula I. It will also be appreciated that suchanhydride polymerization terminating agents are also suitable forterminating the living polymer chain-end of a compound of formula I′, orfreebase thereof. Such anhydride polymerization terminating agentsinclude, but are not limited to, those set forth in Table 7, below.

TABLE 7 Representative Anhydride Polymerization Terminating Agents

A-1 

A-2 

A-3 

A-4 

A-5 

A-6 

A-7 

A-8 

A-9 

A-10

A-11

A-12

A-13

A-14

A-15

A-16

In other embodiments, the R⁴ moiety of the R^(2a) group of formula IIIis incorporated by derivatization of the amino group of formula I′, orfreebase thereof, via reaction with a polymerization terminating agenthaving a suitable leaving group. It will also be appreciated that suchpolymerization terminating agents are also suitable for terminating theliving polymer chain-end of a compound of formula I′, or freebasethereof Examples of these polymerization terminating agents include, butare not limited to, those set forth in Table 8, below.

TABLE 8 Representative Polymerization Terminating Agents

L-1 

L-2 

L-3 

L-4 

L-5 

L-6 

L-7 

L-8 

L-9 

L-10

L-11

L-12

L-13

L-14

L-15

L-16

L-17

L-18

L-19

L-20

L-21

L-22

L-23

L-24

L-25

L-26

L-27

L-28

L-29

L-30

L-31

L-32

L-33

L-34

L-35

L-36

L-37

L-38

L-39

L-40

L-41

L-42wherein each L is a suitable leaving group as defined above and inclasses and subclasses as described above and herein.

In certain embodiments, the hydrophilic polymer block is poly(ethyleneglycol) (PEG) having a terminal amine salt (“PEG macroinitiator”). ThisPEG macroinitiator initiates the polymerization of NCAs to provide themultiblock copolymers of the present invention. Such polymers having aterminal amine salt may be prepared from synthetic polymers having aterminal amine. Such synthetic polymers having a terminal amine groupare known in the art and include PEG-amines. PEG-amines may be obtainedby the deprotection of a suitably protected PEG-amine. Preparation ofsuch suitably protected PEG-amines, and methods of deprotecting thesame, is described in detail in U.S. patent application Ser. No.11/256,735, filed Oct. 24, 2005 the entirety of which is herebyincorporated herein by reference.

As described in U.S. Ser. No. 11/256,735, suitably protected PEG-aminesmay be formed by terminating the living polymer chain end of a PEG witha terminating agent that contains a suitably protected amine. Thesuitably protected amine may then be deprotected to generate a PEG thatis terminated with a free amine that may subsequently be converted intothe corresponding PEG-amine salt macroinitiator. In certain embodiments,the PEG-amine salt macroinitiator of the present invention is prepareddirectly from a suitably protected PEG-amine by deprotecting saidprotected amine with an acid. Accordingly, in other embodiments, theterminating agent has suitably protected amino group wherein theprotecting group is acid-labile.

Alternatively, suitable synthetic polymers having a terminal amine saltmay be prepared from synthetic polymers that contain terminal functionalgroups that may be converted to amine salts by known synthetic routes.In certain embodiments, the conversion of the terminal functional groupsto the amine salts is conducted in a single synthetic step. In otherembodiments, the conversion of the terminal functional groups to theamine salts is achieved by way of a multi-step sequence. Functionalgroup transformations that afford amines, amine salts, or protectedamines are well known in the art and include those described in Larock,R. C., “Comprehensive Organic Transformations,” John Wiley & Sons, NewYork, 1999.

Scheme 3 above shows one exemplary method for preparing the bifunctionalPEGs used to prepare the multiblock copolymers of the present invention.At step (a), the polymerization initiator is treated with a suitablebase to form D. A variety of bases are suitable for the reaction at step(a). Such bases include, but are not limited to, potassiumnaphthalenide, diphenylmethyl potassium, triphenylmethyl potassium, andpotassium hydride. At step (b), the resulting anion is treated withethylene oxide to form the polymer E. Polymer E can be transformed atstep (d) to a compound of formula A directly by terminating the livingpolymer chain-end of E with a suitable polymerization terminator toafford a compound of formula A. Alternatively, polymer E may be quenchedat step (c) to form the hydroxyl compound F. Compound F is thenderivatized to afford a compound of formula A by methods known in theart, including those described herein. Each of the R¹, A, n, and Qgroups depicted in Scheme 3 are as defined and described in classes andsubclasses, singly and in combination, herein.

Although certain exemplary embodiments are depicted and described aboveand herein, it will be appreciated that compounds of the invention canbe prepared according to the methods described generally above usingappropriate starting materials by methods generally available to one ofordinary skill in the art. Additional embodiments are exemplified inmore detail herein.

Methods of preparing micelles are known to one of ordinary skill in theart. Micelles can be prepared by a number of different dissolutionmethods. In the direct dissolution method, the block copolymer is addeddirectly to an aqueous medium with or without heating and micelles arespontaneously formed up dissolution. The dialysis method is often usedwhen micelles are formed from poorly aqueous soluble copolymes. Thecopolymer is dissolved in a water miscible organic solvent such asN-methyl pyrollidinone, dimethylformamide, dimethylsulfoxide,tetrahydrofuran, or dimethylacetamide, and this solution is thendialyzed against water or another aqueous medium. During dialysis,micelle formation is induced and the organic solvent is removed.Alternatively, the block copolymer can be dissolved in in a watermiscible organic solvent such as N-methyl pyrollidinone,dimethylformamide, dimethylsulfoxide, tetrahydrofuran, ordimethylacetamide and added dropwise to water or another aqueous medium.The micelles can then be isolated by filtration or lyophilization.

In one embodiment, drug-loaded miclles possessing carboxylic acidfunctionality in the outer core are crosslinked by addition of zincchloride to the micelle solution along with a small amount of sodiumbicarbonate to neutralize any hydrochloric acid by-product. In thisbasic pH environment, the reaction of zinc chloride with thepoly(aspartic acid) crosslinking block should be rapid and irreversible.

In another embodiment, drug loaded micelles possessing aminefunctionality in the outer core are crosslinked by the addition of abifunctional, or multi-functional aldehyde-containing molecule whichforms pH-reversible imine crosslinks. In another embodiment, drug loadedmicelles possessing aldehyde functionality in the outer core arecrosslinked by the addition of a bifunctional, or multi-functionalamine-containing molecule which forms pH-reversible imine crosslinks.

In another embodiment, drug loaded micelles possessing alcohol or aminefunctionality in the outer core are crosslinked by the addition of abifunctional, or multi-functional carboxylic acid-containing moleculesand a coupling agent to form amide or ester crosslinks. In yet anotherembodiment, drug loaded micelles possessing carboxylic acidfunctionality in the outer core are crosslinked by the addition of abifunctional, or multi-functional amine or alcohol-containing moleculesand a coupling agent to form amide or ester crosslinks. Such couplingagents include, but are not limited to, carbodiimides (e.g.1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), diisopropylcarbodiimide (DIC), dicyclohexyl carbodiimide (DCC)), aminium orphosphonium derivatives (e.g. PyBOP, PyAOP, TBTU, HATU, HBTU), or acombination of 1-hydroxybenzotriazole (HOBt) and a aminium orphosphonium derivative.

In another embodiment, drug loaded micelles possessing aldehyde orketone functionality in the outer core are crosslinked by the additionof a bifunctional, or multifunctional hydrazine or hydrazide-containingmolecule to form pH-reversible hydrazone crosslinks. In still otherembodiments, drug loaded micelles hydrazine or hydrazide-functionalityin the outer core are crosslinked by the addition of a bifunctional, ormultifunctional aldehyde or ketone-containing molecule to formpH-reversible hydrazone crosslinks.

In another embodiment, drug loaded micelles possessing thiolfunctionality in the outer core are crosslinked by the addition of anoxidizing agent (e.g. metal oxides, halogens, oxygen, peroxides, ozone,peroxyacids, etc.) to form disulfide crosslinks. It will be appreciatedthat disulfide crosslinks are reversible in the presence of a suitablereducing agent (e.g. glutathione, dithiothreitol (DTT), etc.).

In yet another embodiment, drug loaded micelles possessing bothcarboxylic acid and thiol functionality in the outer core can be dualcrosslinked by the addition of an oxidizing agent (e.g. metal oxides,halogens, oxygen, peroxides, ozone, peroxyacids, etc.) to form disulfidecrosslinks followed by the addition of zinc chloride to the micellesolution along with a small amount of sodium bicarbonate to neutralizeany hydrochloric acid by-product. It will be appreciated that such adual-crosslinked micelle is reversible only in the presence of acid anda reducing agent (e.g. glutathione, dithiothreitol (DTT), etc.).

According to another aspect, the present invention provides a method forpreparing a micelle comprising a multiblock copolymer which comprises apolymeric hydrophilic block, a crosslinked poly(amino acid block), and apoly(amino acid) block, characterized in that said micelle has an innercore, a crosslinked outer core, and a hydrophilic shell, said methodcomprising the steps of:

-   (a) providing a multiblock copolymer of formula I:

wherein:

n is 10-2500;

m is 1 to 1000;

m′ is 1 to 1000;

R^(x) is a natural or unnatural amino acid side-chain group that iscapable of crosslinking;

R^(y) is a hydrophobic or ionic, natural or unnatural amino acidside-chain group;

R¹ is —Z(CH₂CH₂Y)_(p)(CH₂)_(t)R³, wherein:

-   -   Z is —O—, —S—, —C═C—, or —CH₂—;    -   each Y is independently —O— or —S—;    -   p is 0-10;    -   t is 0-10; and    -   R³ is —N₃, —CN, a mono-protected amine, a di-protected amine, a        protected aldehyde, a protected hydroxyl, a protected carboxylic        acid, a protected thiol, a 9-30 membered crown ether, or an        optionally substituted group selected from aliphatic, a 5-8        membered saturated, partially unsaturated, or aryl ring having        0-4 heteroatoms independently selected from nitrogen, oxygen, or        sulfur, an 8-10 membered saturated, partially unsaturated, or        aryl bicyclic ring having 0-5 heteroatoms independently selected        from nitrogen, oxygen, or sulfur, or a detectable moiety;

Q is a valence bond or a bivalent, saturated or unsaturated, straight orbranched C₁₋₁₂ alkylene chain, wherein 0-6 methylene units of Q areindependently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—,—C(O)—, —SO—, —SO₂—, —NHSO₂—, —SO₂NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or—NHC(O)O—, wherein:

-   -   -Cy- is an optionally substituted 5-8 membered bivalent,        saturated, partially unsaturated, or aryl ring having 0-4        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, or an optionally substituted 8-10 membered bivalent        saturated, partially unsaturated, or aryl bicyclic ring having        0-5 heteroatoms independently selected from nitrogen, oxygen, or        sulfur;

R^(2a) is a mono-protected amine, a di-protected amine, —N(R⁴)₂,—NR⁴C(O)R⁴, —NR⁴C(O)N(R⁴)₂, —NR⁴C(O)OR⁴, or —NR⁴SO₂R⁴; and

each R⁴ is independently an optionally substituted group selected fromhydrogen, aliphatic, a 5-8 membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, oraryl bicyclic ring having 0-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or a detectable moiety, or:

-   -   two R⁴ on the same nitrogen atom are taken together with said        nitrogen atom to form an optionally substituted 4-7 membered        saturated, partially unsaturated, or aryl ring having 1-4        heteroatoms independently selected from nitrogen, oxygen, or        sulfur,

-   (b) combining said compound of formula I with a therapeutic agent;    and

-   (c) treating the resulting micelle with a crosslinking reagent to    crosslink R^(x).

In one embodiment, drugs are loaded into the micelle inner core byadding an aliquot of a copolymer solution in water to the drug to beincorporated. For example, a stock solution of the drug in a polarorganic solvent is made and allowed to evaporate, and then thecopolymer/water solution is added. In another embodiment, the drug isincorporated using an oil in water emulsion technique. In this case, thedrug is dissolved in an organic solvent and added dropwise to themicelle solution in water, and the drug is incorporated into the micelleduring solvent evaporation. In another embodiment, the drug is dissolvedwith the copolymer in a common polar organic solvent and dialyzedagainst water or another aqueous medium. See Allen, C.; Maysinger, D.;Eisenberg A. Colloid Surface B 1999, 16, 3-27.

In still another embodiment, the loading and crosslinking of drug-filledmicelles is carried out by dissolving neutral doxorubicin, camptothecin,or paclitaxel and the block copolymer in a polar solvent such as acetoneor ethanol, followed by slow addition to water or buffer solution. Dueto the limited solubility of DOX and CPT in water, the drug is forcedinto the core of the micelle, effectively encapsulating the drug.

5. Uses, Methods, and Compositions

As described herein, micelles of the present invention can encapsulate awide variety of therapeutic agents useful for treating a wide variety ofdiseases. In certain embodiments, the present invention provides adrug-loaded micelle, as described herein, wherein said micelle is usefulfor treating the disorder for which the drug is known to treat.According to one embodiment, the present invention provides a method fortreating one or more disorders selected from pain, inflammation,arrhythmia, arthritis (rheumatoid or osteoarthritis), atherosclerosis,restenosis, bacterial infection, viral infection, depression, diabetes,epilepsy, fungal infection, gout, hypertension, malaria, migraine,cancer or other proliferative disorder, erectile dysfunction, a thyroiddisorder, neurological disorders and hormone-related diseases,Parkinson's disease, Huntington's disease, Alzheimer's disease, agastro-intestinal disorder, allergy, an autoimmune disorder, such asasthma or psoriasis, osteoporosis, obesity and comorbidities, acognitive disorder, stroke, AIDS-associated dementia, amyotrophiclateral sclerosis (ALS, Lou Gehrig's disease), multiple sclerosis (MS),schizophrenia, anxiety, bipolar disorder, tauopothy, a spinal cord orperipheral nerve injury, myocardial infarction, cardiomyocytehypertrophy, glaucoma, an attention deficit disorder (ADD or ADHD), asleep disorder, reperfusion/ischemia, an angiogenic disorder, or urinaryincontinence, comprising administering to a patient a micelle comprisinga multiblock copolymer which comprises a polymeric hydrophilic block, acrosslinked poly(amino acid block), and a poly(amino acid block),characterized in that said micelle has a drug-loaded inner core, acrosslinked outer core, and a hydrophilic shell, wherein said micelleencapsulates a therapeutic agent suitable for treating said disorder.

In other embodiments, the present invention provides a method fortreating one or more disorders selected from autoimmune disease, aninflammatory disease, a metabolic disorder, a psychiatric disorder,diabetes, an angiogenic disorder, tauopothy, a neurological orneurodegenerative disorder, a spinal cord injury, glaucoma, baldness, ora cardiovascular disease, comprising administering to a patient amicelle comprising a multiblock copolymer which comprises a polymerichydrophilic block, a crosslinked poly(amino acid block), and apoly(amino acid block), characterized in that said micelle has adrug-loaded inner core, a crosslinked outer core, and a hydrophilicshell, wherein said micelle encapsulates a therapeutic agent suitablefor treating said disorder.

In certain embodiments, drug-loaded micelles of the present inventionare useful for treating cancer. Accordingly, another aspect of thepresent invention provides a method for treating cancer in a patientcomprising administering to a patient a micelle comprising a multiblockcopolymer which comprises a polymeric hydrophilic block, a crosslinkedpoly(amino acid block), and a poly(amino acid block), characterized inthat said micelle has a drug-loaded inner core, a crosslinked outercore, and a hydrophilic shell, wherein said micelle encapsulates achemotherapeutic agent. According to another embodiment, the presentinvention relates to a method of treating a cancer selected from breast,ovary, cervix, prostate, testis, genitourinary tract, esophagus, larynx,glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma, lung,epidermoid carcinoma, large cell carcinoma, small cell carcinoma, lungadenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma, thyroid,follicular carcinoma, undifferentiated carcinoma, papillary carcinoma,seminoma, melanoma, sarcoma, bladder carcinoma, liver carcinoma andbiliary passages, kidney carcinoma, myeloid disorders, lymphoiddisorders, Hodgkin's, hairy cells, buccal cavity and pharynx (oral),lip, tongue, mouth, pharynx, small intestine, colon-rectum, largeintestine, rectum, brain and central nervous system, and leukemia,comprising administering a micelle in accordance with the presentinvention wherein said micelle encapsulates a chemotherapeutic agentsuitable for treating said cancer.

P-glycoprotein (Pgp, also called multidrug resistance protein) is foundin the plasma membrane of higher eukaryotes where it is responsible forATP hydrolysis-driven export of hydrophobic molecules. In animals, Pgpplays an important role in excretion of and protection fromenvironmental toxins; when expressed in the plasma membrane of cancercells, it can lead to failure of chemotherapy by preventing thehydrophobic chemotherapeutic drugs from reaching their targets insidecells. Indeed, Pgp is known to transport hydrophobic chemotherapeuticdrugs out of tumor cells. According to one aspect, the present inventionprovides a method for delivering a hydrophobic chemotherapeutic drug toa cancer cell while preventing, or lessening, Pgp excretion of thatchemotherapeutic drug, comprising administering a drug-loaded micellecomprising a multiblock polymer of the present invention loaded with ahydrophobic chemotherapeutic drug. Such hydrophobic chemotherapeuticdrugs are well known in the art and include those described herein.

Compositions

According to another embodiment, the invention provides a compositioncomprising a micelle of this invention or a pharmaceutically acceptablederivative thereof and a pharmaceutically acceptable carrier, adjuvant,or vehicle. In certain embodiments, the composition of this invention isformulated for administration to a patient in need of such composition.In other embodiments, the composition of this invention is formulatedfor oral administration to a patient.

The term “patient”, as used herein, means an animal, preferably amammal, and most preferably a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts.

Salts derived from appropriate bases include alkali metal (e.g., sodiumand potassium), alkaline earth metal (e.g., magnesium), ammonium andN+(C1-4 alkyl)4 salts. This invention also envisions the quaternizationof any basic nitrogen-containing groups of the compounds disclosedherein. Water or oil-soluble or dispersible products may be obtained bysuch quaternization.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

The pharmaceutically acceptable compositions of this invention may beorally administered in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added. In certain embodiments,pharmaceutically acceptable compositions of the present invention areenterically coated.

Alternatively, the pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

The pharmaceutically acceptable compositions of this invention may alsobe administered topically, especially when the target of treatmentincludes areas or organs readily accessible by topical application,including diseases of the eye, the skin, or the lower intestinal tract.Suitable topical formulations are readily prepared for each of theseareas or organs.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutically acceptable compositionsmay be formulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutically acceptable compositions canbe formulated in a suitable lotion or cream containing the activecomponents suspended or dissolved in one or more pharmaceuticallyacceptable carriers. Suitable carriers include, but are not limited to,mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutically acceptable compositions may beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the pharmaceuticallyacceptable compositions may be formulated in an ointment such aspetrolatum.

The pharmaceutically acceptable compositions of this invention may alsobe administered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

In certain embodiments, the pharmaceutically acceptable compositions ofthis invention are formulated for oral administration.

The amount of the compounds of the present invention that may becombined with the carrier materials to produce a composition in a singledosage form will vary depending upon the host treated, the particularmode of administration. Preferably, the compositions should beformulated so that a dosage of between 0.01-100 mg/kg body weight/day ofthe drug can be administered to a patient receiving these compositions.

It will be appreciated that dosages typically employed for theencapsulated drug are contemplated by the present invention. In certainembodiments, a patient is administered a drug-loaded micelle of thepresent invention wherein the dosage of the drug is equivalent to whatis typically administered for that drug. In other embodiments, a patientis administered a drug-loaded micelle of the present invention whereinthe dosage of the drug is lower than is typically administered for thatdrug.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It will be understoodthat these examples are for illustrative purposes only and are not to beconstrued as limiting this invention in any manner.

Examples Preparation of Bifunctional PEGs and Multiblock Copolymers ofthe Present Invention

As described generally above, multiblock copolymers of the presentinvention are prepared using the heterobifunctional PEGs describedherein and in U.S. patent application Ser. No. 11/256,735, filed Oct.24, 2005, the entirety of which is hereby incorporated herein byreference. The preparation of multiblock polymers in accordance with thepresent invention is accomplished by methods known in the art, includingthose described in detail in U.S. patent application Ser. No.11/325,020, filed Jan. 4, 2006, the entirety of which is herebyincorporated herein by reference.

Example 1

Dibenzylamino-poly(ethylene glycol)-t-butyldiphenylsilylpropene: To astirred solution of dibenzylaminoethanol (482 mg, 2 mmol) in anhydrousTHF (200 mL) was added a solution of potassium naphthalenide in THF (0.2M, 10 mL, 2 mmol). The resulting solution was cooled to 0° C., thenethylene oxide (20 g, 454 mmol) was introduced to the alkoxide solutionusing Schlenk techniques. Upon complete addition of the ethylene oxide,the flask was backfilled with Argon, sealed and stirred at 40° C. After24 h, t-butyldiphenylsilylpropargyl bromide (3.54 g, 10 mmol) was addedto the reaction using Schlenk techniques. The solution was stirred forand additional 12 h at 40° C., allowed to cool, and the solvent removed.The resulting viscous liquid was purified by solid phase extraction (Theliquid was loaded onto 400 mL silica gel which was rinsed with 3% MeOHin CHCl₃ (1 L) followed by 10% MeOH in CHCl₃ (1 L) which containe thepolymer product) then precipitation into cold diethyl ether to give awhite powder (14.4 g, 72% yield). ¹H NMR (400 MHz, DMSO-d₆, δ) 7.8-7.2(m, Ar—H), 4.39 (s, CH₂-alkyne), 3.7-3.3 (br-m, —O—CH₂—CH₂—) 1.03 (s,t-butyl). Mn˜9800 by ¹H NMR.

Amino-poly(ethylene glycol)-t-butyldiphenylsilylpropene: To a 100 mLround bottom flask was added 10% palladium hydroxide on carbon (0.2 g)and methanol (200 mL). Dibenzylamino-poly(ethyleneglycol)-t-butyldiphenylsilylpropene (2 g) and ammonium formate (2 g) wasadded and the reaction heated to reflux. After 6 hours, potassiumcarbonate (4 g) was added and the solution stirred for an additional 3hours at reflux. The solution was diluted with chloroform (200 mL),allowed to cool, then filtered over basic alumina. The solvent wasevaporated and the polymer product precipitated into cold diethyl etherand recovered as a white powder following filtration (1.2 g, 60% yield).¹H NMR (400 MHz, DMSO-d₆, δ) 7.6-7.3 (m, Ar—H), 4.19 (s, CH₂-alkyne),3.7-3.3 (br-m, —O—CH₂—CH₂—O—), 0.96 (s, t-butyl).

Ammonium chloride-poly(ethylene glycol)-t-butyldiphenylsilylpropene: Toa 50 mL round bottom flask with stir bar was added amino-poly(ethyleneglycol)-t-butyldiphenylsilylpropene (1.2 g, 0.1 mmol) and THF (5 mL).The solution was stirred at room temperature until a homogeneoussolution was present. 4 M HCl in dioxane (5 mL) was then added and thesolution stirred for 1 hour. The polymer was precipitated into coldether to give a white powder (1 g, 83% yield). ¹H NMR (400 MHz, DMSO-d₆,δ) 7.85 (br-s, —NH₃Cl, 7.58 (m, Ar—H), 7.45 (m, Ar—H), 7.41 (m, Ar—H),4.17 (s, CH₂-alkyne), 3.7-3.3 (br-m, —O—CH₂—CH₂—), 0.97 (s, t-butyl).

TBDPS-propyne-poly(ethylene glycol)-b-poly(t-butyl asparticacid)-b-[poly(phenylalanine)-co-poly(t-butyl tyrosine)]: To a 100 mLreaction vessel equipped with glass stir bar and Teflon valve was addedammonium chloride-poly(ethylene glycol)-t-butyldiphenylsilylpropene (0.6g, 0.05 mmol) and t-butyl aspartic acid NCA (0.11 g, 0.5 mmol). Theflask was evacuated for 1 h then backfilled with Ar. Anhydrous NMP (7mL) was added via syringe then the flask sealed under and Ar atmosphereand stirred at 80° C. After 48 h, phenylalanine NCA (0.32 g, 1.2 mmol)and t-butyl tyrosine (0.08 g, 0.3 mmol) were dried under vacuum,dissolved in anhydrous NMP (4 mL), and added to the reaction solutionusing Schlenk techniques. The resulting solution was stirred at 80° C.for an additional 48 h. The polymerization was then allowed to cool andthe product precipitated into cold ether, giving a white powder (0.7 g,65%). ¹H NMR (400 MHz, DMSO-d₆, δ) 9.10, 8.04, 7.56, 7.41, 7.14, 6.95,6.60, 4.51, 3.7-3.2, 2.92, 2.70, 1.37, 1.31, 0.97. Mn˜14,500 by ¹H NMR.

TBDPS-propyne-poly(ethylene glycol)-b-poly(asparticacid)-b-[poly(phenylalanine)-co-poly(tyrosine)]: To a 50 mL round bottomflask with stir bar was added TBDPS-propyne-poly(ethyleneglycol)-b-poly(t-butyl asparticacid)-b-[poly(phenylalanine)-co-poly(t-butyl tyrosine)] (0.5 g) andformic acid (10 mL). The solution was stirred at 50° C. for 12 h, thenthe solvent evaporated. The residue was dissolved in methanol and thesolvent again evaporated. The residue was again dissolved in methanolthen precipitated into cold ether, giving a white powder (0.4 g, 80%yield). ¹H NMR (400 MHz, DMSO-d₆, δ) 9.17, 8.14, 8.05, 7.56, 7.41, 7.21,7.15, 6.96, 6.60, 4.51, 3.7-3.2, 2.93, 2.71, 0.97.

Example 2

Dibenzylamino-polyethylene glycol-alcohol: To a stirred solution ofdibenzylaminoethanol (242 mg, 1 mmol) in anhydrous THF (100 mL) wasadded a solution of potassium naphthalenide in THF (0.2 M, 1 mL, 1mmol). The resulting solution was stirred for 5 minutes then cooled to0° C. Ethylene oxide (10 g, 227 mmol) was introduced to the alkoxidesolution using Schlenk techniques. Upon complete addition of theethylene oxide, the flask was backfilled with Argon, sealed and stirredat 40° C. for 24 h. The reaction was quenched with water (1 mL) followedby the removal of solvent under reduced pressure. The resulting viscousliquid was purified by solid phase extraction (The liquid was loadedonto 200 mL silica gel which was rinsed with 3% MeOH in CHCl₃ (1 L)followed by 10% MeOH in CHCl₃ (1 L) which contained the polymer product)then precipitation into cold diethyl ether to give a white powder (6.8g, 68% yield). NMR (400 MHz, DMSO-d₆, δ) 7.4-7.2 (m, Ar—H), 4.63 (t,CH₂OH), 3.7-3.3 (br-m, —O—CH₂—CH₂—). GPC (DMF, PEG standards)M_(n)=7,300; PDI=1.03.

BOC-amino-poly(ethylene glycol)-alcohol: To a 250 mL round bottom flaskwas added 10% palladium hydroxide on carbon (1 g) and methanol (100 mL).Dibenzylamino-poly(ethylene glycol)-alcohol (5 g) and ammonium formate(5 g) was added and the reaction heated to reflux. After 6 hours,potassium carbonate (10 g) was added and the solution stirred for anadditional 3 hours at reflux. The solution was diluted with chloroform(300 mL), allowed to cool, then filtered over basic alumina. The solventwas evaporated and the polymer redissolved in methanol (100 mL). BOCanhydride (3 g) and DMAP (1 g) were added and the solution stirred atroom temperature for 12 h. The solvent was removed and the residue waspurified by solid phase extraction (The liquid was loaded onto 200 mLsilica gel which was rinsed with 3% MeOH in CHCl₃ (1 L) followed by 10%MeOH in CHCl₃ (1 L) which contained the polymer product) thenprecipitation into cold diethyl ether to give a white powder (4.2 g, 84%yield). ¹H NMR (400 MHz, DMSO-d₆, δ) 6.82 (br-s, CH₂—NH—CO—), 4.63 (t,CH₂OH), 3.7-3.3 (br-m, —O—CH₂—CH₂—O—), 1.40 (s, —C—(CH₃)₃).

BOC-amino-poly(ethylene glycol)-aryl-propyne: To a 50 mL round bottomflask with stir bar was added propargyl phenol (0.37 g, 2.5) mmol),triphenylphosphine (0.53 g, 2 mmol), BOC-amino-poly(ethyleneglycol)-alcohol (3.6 g, 0.5 mmol) and THF (10 mL). The reaction wasstirred at room temperature until a homogeneous solution was presentthen DIAD (0.3 g, 1.5 mmol) was added and the reaction stirred at roomtemperature for 16 hours. The solvent was then removed under reducedpressure and the resulting viscous liquid was purified by solid phaseextraction (The liquid was loaded onto 200 mL silica gel which wasrinsed with 3% MeOH in CHCl₃ (1 L) followed by 10% MeOH in CHCl₃ (1 L)which contained the polymer product). Pure product was obtained as awhite powder following precipitation into cold ether (2.8 g, 77% yield).¹H NMR (400 MHz, DMSO-d₆, δ) 6.92 (m, Ar—H), 4.68 (s, O—CH₂-alkyne),4.04 (s, Ar—O—CH₂), 3.7-3.3 (br-m, —O—CH₂—CH₂—), 2.55 (t, alkyne-H),1.42 (s, —C—(CH₃)₃).

Propyne-aryl-poly(ethylene glycol)-ammonium chloride: To a 50 mL roundbottom flask with stir bar was added BOC-amino-poly(ethyleneglycol)-aryl-propyne (2 g, 0.1 mmol) and THF (5 mL). The solution wasstirred at room temperature until a homogeneous solution was present. 4M HCl in dioxane (5 mL) was then added and the solution stirred for 2hours. The polymer was precipitated into cold ether to give a whitepowder (1.7 g, 85% yield). ¹H NMR (400 MHz, DMSO-d₆, δ) 7.76 (br-s,—NH₃Cl), 6.90 (s, Ar—H), 4.71 (s, O—CH₂-alkyne), 4.02 (s, Ar—O—CH₂),3.7-3.3 (br-m, —O—CH₂—CH₂—).

Propyne-aryl-poly(ethylene glycol)-b-poly(t-butyl asparticacid)-b-[poly(phenylalanine)-co-poly(t-butyl tyrosine)]: To a 100 mLreaction vessel equipped with glass stir bar and Teflon valve was addedPropyne-aryl-poly(ethylene glycol)-ammonium chloride (1.46 g, 0.2 mmol)and t-butyl aspartic acid NCA (0.43 g, 2 mmol). The flask was evacuatedfor 1 h then backfilled with Ar. Anhydrous NMP (20 mL) was added viasyringe then the flask sealed under and Ar atmosphere and stirred at 80°C. After 48 h, phenylalanine NCA (1.26 g, 4.8 mmol) and t-butyl tyrosine(0.3 g, 1.2 mmol) were dried under vacuum, dissolved in anhydrous NMP(15 mL), and added to the reaction solution using Schlenk techniques.The resulting solution was stirred at 80° C. for an additional 48 h. Thepolymerization was then allowed to cool and the product precipitatedinto cold ether, giving a white powder (1.6 g, 54%). ^(I)ll NMR (400MHz, DMSO-d₆, δ) 8.16, 8.08, 7.95, 7.21, 7.16, 6.91, 6.67, 4.70, 4.52,4.02, 3.7-3.2, 3.04, 2.69, 2.19, 1.91, 1.37. Mn˜11,600 by ¹H NMR.

Propyne-aryl-poly(ethylene glycol)-b-poly(asparticacid)-b-[poly(phenylalanine)-co-poly(tyrosine)]: To a 50 mL round bottomflask with stir bar was added Propyne-aryl-poly(ethyleneglycol)-b-poly(t-butyl asparticacid)-b-[poly(phenylalanine)-co-poly(t-butyl tyrosine)] (1 g) and formicacid (10 mL). The solution was stirred at 50° C. for 12 h, then thesolvent evaporated. The residue was dissolved in methanol and thesolvent again evaporated. The residue was again dissolved in methanolthen precipitated into cold ether, giving a white powder. ¹H NMR (400MHz, DMSO-d₆, δ) 8.13, 8.07, 7.21, 7.18, 7.15, 7.00, 6.87, 6.60, 4.71,4.52, 4.02, 3.7-3.2, 2.94, 2.74.

Example 3

CMC Calculations: The CMC of micelles prepared from multiblockcopolymers were determined using the method described by Eisnberg.(Astafieva, I.; Zhong, X. F.; Eisenberg, A. “Critical MicellizationPhenomena in Block Copolymer Polyelectrolyte Solutions” Macromolecules1993, 26, 7339-7352.) To perform these experiments, a constantconcentration of pyrene (5×10⁻⁷ M) was equilibrated with varyingconcentrations of block copolymer (5×10⁻⁴ M to 1×10⁻⁸ M) in water at 50°C. for 2 hours, then stirred overnight. Examination of each sample'sfluorescence excitation spectra (excited at 390 nm) revealed whether thepyrene was encapsulated in the diblock copolymer micelle (λ_(max)=338nm) or free in aqueous solution (λ_(max)=333 nm). Plotting the ratio ofthe intensities between 338nm and 333nm (I₃₃₈/I₃₃₃) vs. log of the blockcopolymer concentration allows for the graphical interpretation of theCMC value. In these experiments, I₃₃₈/I₃₃₃ values of 1.3-1.5 representpyrene encapsulated in block copolymer micelles and I₃₃₅/I₃₃₃ values of0.5-0.6 correspond to pyrene free in solution (no micelles are present).CMC experimental data for propyne-aryl-poly(ethyleneglycol)-b-poly(aspartic acid)-b-[poly(phenylalanine)-co-poly(tyrosine)](shown below) afforded a CMC value of 5×10⁻⁶ M. See FIG. 11.

Example 4

Preparation of zinc crosslinked micelles with encapsulated pyrene:Crosslinked micelles containing encapsulated pyrene were prepared bystirring pyrene and propyne-aryl-poly(ethylene glycol)-b-poly(asparticacid)-b-[poly(phenylalanine)-co-poly(tyrosine)] in an aqueous zincchloride solution at 50° C. for two hours then an additional 16 hours atroom temperature (2.5×10⁻⁴ M polymer, 0.5 M ZnCl₂, 5×10⁻⁷M pyrene). 2 mLof 0.5 M NaHCO₃ was added, raising the pH to 8.2 from 3.1, and resultingsolution was allowed to stir for an additional 2 hours. The solution wasdiluted to give samples with polymer concentrations of 1×10⁻⁴, 5×10⁻⁵,1×10⁻⁵, 5×10⁻⁶ M. Examination of each sample's fluorescence excitationspectra (excited at 390 nm) revealed whether the pyrene was encapsulatedin the diblock copolymer micelle (λ_(max)=338 nm) or free in aqueoussolution (λ_(max)=333 nm). Plotting the ratio of the intensities between338nm and 333nm (I₃₃₈/I₃₃₃) vs. log of the block copolymer concentrationallows for the graphical interpretation of the CMC value. In theseexperiments, I₃₃₈/I₃₃₃ values of 1.3-1.5 represent pyrene encapsulatedin block copolymer micelles and I₃₃₈/I₃₃₃ values of 0.5-0.6 correspondto pyrene free in solution (no micelles are present). A controlexperiment was performed in the absence of zinc chloride to show theeffect of dilution on uncrosslinked micelles. Comparison between thezinc crosslinked micelles and uncrosslinked control experiments is shownin FIG. 12.

Example 5

Reversing of Zinc Crosslink by pH Adjustment: The pyrene loadedcrosslinked micelles were uncrosslinked with the addition of acid. Forthese experiments, lactic acid (100 uL) was added to each of thecrosslinked micelle samples and the fluorescence excitation spectra ofpyrene recorded. The pH of the samples was lowered to pH 3.1 after theaddition of the lactic acid (from 8.2 for the crosslinked micelles).Graph of the pyrene loaded crosslinked micelles before and after theaddition of lactic acid is shown in FIG. 13.

Example 6

Example 7

Drug Loading and Crosslinking: Experimentally, the loading andcrosslinking of drug-filled micelles is carried out by dissolvingneutral doxorubicin and the block copolymer in a polar solvent such asethanol, followed by slow addition to water or buffer solution. Due tothe limited solubility of DOX and CPT in water, the drug is forced intothe core of the micelle, effectively encapsulating the drug.Crosslinking is achieved by addition of zinc chloride to the micellesolution along with a small amount of sodium bicarbonate to neutralizeany hydrochloric acid by-product. In this basic pH environment, thereaction of zinc chloride with the poly(aspartic acid) crosslinkingblock is rapid and irreversible. The crosslinked nanovectors areisolated using ultrafiltration membranes (Amicon Ultracel YM-30membranes) and subsequently lyophilized before storage and/orcharacterization. Unencapsulated drug is removed by the ultrafiltrationprocess, and drug-loading is quantified by dissolving known quantitiesof the drug-loaded, micelle powder in DMF and quantifying drugconcentration by UV-Vis spectroscopy based on a previously determinedextinction coefficients (g for neutral DOX and CPT (DOX absorbance at485 nm, CPT absorbance at 370 nm).

Drug-loaded, polymer micelles are characterized before and after zincchloride addition to confirm the effectiveness of crosslinking and toquantify the pH at which micelle dissociation occurs. Fluorescencespectroscopy is an appropriate method to determine drug release fromcrosslinked polymer micelles in response to environmental changes sincethe fluorescence of micelle encapsulated DOX and CPT is negligible dueto self-quenching in the micelle core but is highly fluorescent in itsfree form. After reversible crosslinking, qualitative fluorescenceexperiments are performed to confirm effective crosslinking andstability of drug-loaded micelles. For example, crosslinked micelles aretreated with sodium dodecyl sulfate (SDS), a common surfactant known todisrupt uncrosslinked micelles. After treatment with SDS, drug-filledmicelles, both crosslinked and uncrosslinked, are evaluated usingfluorescence spectroscopy to detect the presence of released DOX(excitation at 485 nm, emission at 590 nm) or CPT (excitation at 370 nm,emission at 432 nm). In the case of uncrosslinked micelles, fluorescencearising from free DOX or CPT in water is be observed due to SDS-inducedmicelle dissociation.

Example 8 Fluorescence Assay

In another set of experiments, samples of each of crosslinked polymermicelles, uncrosslinked polymer micelles, and free doxorubicin areincubated separately at 37° C. in Dulbecco's Modified Eagle's Medium(DMEM) supplemented with 10% fetal bovine serum (FBS) for 24 hours. Thiscommonly used cell growth medium is used to simulate the ion and pHenvironment encountered at physiological conditions. The concentrationof DOX or CPT in the three samples are adjusted to an equivalent value(e.g. 100 μg/mL) and evaluated for dilution stability using fluorescencespectroscopy. In these experiments, all three samples are diluted toconcentrations below the CMC of the polymer micelles (determined usingpyrene fluorescence experiments described below). Fluorescence should beobserved for the free DOX sample and DOX released from the dissociated,uncrosslinked micelles. If metal-mediated crosslinking is successful,fluorescence from the crosslinked micelles should not be observed,indicating enhanced micelle stability and limited drug release.

Quantitative, acid titration experiments is performed on crosslinked,drug-loaded micelles to determine precise pH values or the pH range atwhich reversible micelle crosslinking occurs. These titrationexperiments are carried out by measuring fluorescence (reported inrelative fluorescence units) at pH values ranging from˜7.4 (DMEM withserum) to 4.0. The pH of the DOX-loaded, crosslinked micelle solution isadjusted by the incremental addition of lactic acid or hydrochloric acid(HCl) and is measured directly in a stirred, fluorescence cell using asmall pH probe (Lazar Ultra-M micro pH electrode). Titration curves areconstructed by plotting fluorescence versus solution pH, and the pH atwhich reversible crosslinking occurs is determined by extrapolation,similar to the pyrene-CMC experiments described in Item D. Utilizingdilution volumes which are below the copolymer CMC values is requiredsince rapid micelle dissociation and drug release, as determined by anincrease in quantum yield, are required to quantify pH reversibility.Titration experiments are repeated with crosslinked micelles made withvarious zinc chloride/block copolymer ratios with the ultimate goal ofdetermining which micellar formulations undergo rapid dissociation at pH6.8 (solid tumor microenvironment) and pH 5.0-5.5 (endosomalcompartments). Fluorescence experiments analyzing the change in quantumyield of the free drug versus pH (control experiment) will also beundertaken to minimize the likelihood of a false positive result.Identical experiments, using encapsulated camptothecin and free CPT, arerepeated for successful formulations to determine whether pH-sensitivedissociation and release varies with different encapsulated drugs. Weanticipate that reversible crosslinking is independent of the drugutilized, permitting the use of a wide range of hydrophobic drugs. It isunderstood that carboxylic acid-containing therapeutics may requireadditional considerations when used in conjunction with zinc-mediatedcrosslinking.

In addition to fluorescence experiments, light scattering analysis ofpolymer micelles is performed to determine both micelle size andmorphology, which are important to future pharmacokinetic andbiodistribution studies. The polymer micelles are analyzed by dynamicand static light scattering experiments at 37° C. in phosphate buffersolution to determine average micelle size (e.g. hydrodynamic radius(Rh)) and micelle size distribution before and after drug loading. Theratio of radius of gyration (Rg), obtained by static light scattering,to Rh offers important information about particle morphology (i.e.spherical micelle, worm-like micelle, vesicle, etc.) before and aftercrosslinking reactions.

Example 9 In Vitro Studies of Cancer-Responsive, Drug-Loaded Micelles

In vitro testing of block copolymer micelles and drug-loaded nanovectorsprovides direct feedback on both the cellular uptake of nanovectors andpotential toxicity of the block copolymers, crosslinking reagents, andthe by-products of the reversible crosslinking reaction. Utilizing theinherent fluorescence of both doxorubicin and camptothecin as well asother common dyes, cellular uptake is monitored and trafficking ofdrug-filled and dye-filled nanovectors in cells lines such as MCF-7(breast cancer), DOX-resistant MCF-7 (breast cancer), HeLa (cervicalcancer), HepG2 (liver cancer), and Chinese hamster ovary (control) usingconfocal laser scanning microscopy. In addition, cell viability studiesare performed comparing micelle-encapsulated forms of CPT and DOX to thefree drugs in the five cell lines mentioned above.

Cellular Uptake of Nanovectors and Release of Small MoleculeTherapeutics In Vitro

Fluorescence microscopy is a frequently used method to investigate theinteractions and intracellular fate of nanoparticulate drug carriers,such as liposomes and micelles, within target cells. To evaluate theuptake and cellular trafficking of cancer-responsive nanovectors,micelles are prepared with encapsulated dyes that fluoresce inside themicelle (Oregon Green or Cy5) and monitored by CLSM. Small amounts ofCy5 are incorporated due to its high quantum yield and its tendency toself-quench at high concentrations in the micelle core. The dye-loadedmicelles are evaluated in MCF-7 (ATCC, HTB-22™), DOX-resistant MCF-7(prepared according to literature protocol), HeLa (ATCC, CCL-2™), HepG2(ATCC, HB-8065™), and Chinese hamster ovary (ATCC, CCL-61™) cell linesto assay the background rate of uptake of crosslinked micelles bypinocytosis. Unless otherwise stated, HeLa, HepG2, and Chinese hamsterovary cells are grown in DMEM supplemented with 10% fetal bovine serum(FBS). MCF-7 and DOX-resistant MCF-7 cells are grown in Roswell ParkMemorial Institute (RPMI) media with 10% heat-inactivated FBS. Forconfocal studies, cells are incubated in the presence of bothnon-loaded, crosslinked micelles (to establish any backgroundfluorescence) and dye-filled, crosslinked micelles (dye—1 ug/mL)directly on cover slips to a confluence of 60-70%, incubated withfluorescent nanovectors for 0.5, 1, or 4 hours, and mounted on glassslides using a fluorophore-free mounting medium. In the case of MCF-7and DOX-resistant MCF-7 cells, the cells are washed three times with PBSpH 7.4 and then fixed to the cover slips with a 1% formaldehyde solutionin PBS for 10 minutes prior to mounting on the glass slides. Since bothDOX and CPT require cellular entry and cytoplasmic delivery to be oftherapeutic value, these basic uptake studies in a variety of cellcultures are an important benchmark in determining their potentialclinical usefulness. In general, micelles with diameters of 100 nm orless are taken up by cells, albeit indiscriminately, by pinocytosis.However, recent studies have shown that drug-loaded nanoparticles alsoundergo rapid exocytosis if endosomal escape is not achieved, and thisphenomenon ultimately results in the reduced efficacy of theencapsulated drug. Notwithstanding this phenomenon, these same studiessuggest that targeted nanovectors, which undergo uptake byreceptor-mediated endocytosis (RME), are more apt to avoid exocytosisand may enter the cell through a different intracellular pathway.⁷⁸

Preliminary studies involving cell-targeted nanovectors are undertakento compare micelle uptake by pinocytosis and RME. To accomplish this,acetylene-functionalized micelles are conjugated with azide-containingfolate or an azide-containing GRGDS oligopeptide by click chemistry asshown in FIG. 14. Relative to normal cells and tissue, the folatereceptor is over-expressed in many epithelial malignancies, such asovarian, colorectal, and breast cancer and has been identified as atumor marker. RGD-containing oligopeptides have been shown to bind toα_(v)β₃ integrins which are over-expressed on actively proliferatingendothelium around cancerous tissue. Both targeting groups have beenused extensively in drug delivery systems and have been shown to promotecellular uptake by cancer cells. Cellular uptake and distribution ofcell-targeted, dye-filled micelles are evaluated in the five previouslydescribed cell lines. Previous studies involving folate-conjugatedpolymer micelles suggest that receptor-mediated endocytosis is a moreefficient method of cellular entry when compared to simple pinocytosis.Micelle formulations which achieve the highest levels of cellular entry,as judged by intracellular fluorescence, are deemed the most promising.Using the click conjugation approach, a range of azide-functional,cell-targeting moieties are attached, making this approach advantageousfor quickly evaluating multiple targeting groups in multiple differentcell lines.

Cellular uptake experiments using dye-filled or dye-conjugated,crosslinked nanovectors are complemented with other confocal studiesinvolving doxorubicin and camptothecin-filled micelles, which provide asignal indicating both uptake and release. As previously discussed, DOXand CPT are inherently fluorescent, but their quantum yield issignificantly reduced in the micelle core due to self quenching.Utilizing this feature, drug distribution analysis of DOX- andCPT-loaded micelles are performed in the five previously described celllines using confocal microscopy. Each cell line is incubated for 2 to 24hours with no DOX or CPT (control), free DOX or CPT (1 μM), anddrug-loaded micelles (both uncrosslinked and zinc-crosslinked, micelleconcentration adjusted to achieve 1 μM DOX and CPT). The nucleus of thecells is stained with Hoechst 33342 (Molecular Probes), and the culturemedia is replaced with phosphate buffer solution prior to confocalmicroscopy. DOX and CPT are topoisomerase inhibitors and require accessto the nucleus to achieve therapeutic effects.

If the drug-loaded micelles are taken up by cells and can escapeendosomal compartments, then fluorescence from DOX and CPT should beobserved in both the cytoplasm and the nucleus of the cell. Successfulmicelle formulations will result in high concentrations of the releaseddrugs in the cell nucleus. Special observation is made in experimentsinvolving folate-targeted micelles and non-targeted micelles inDOX-resistant MCF-7 cells. Drug-resistant cancer cells haveover-expressed proteins which minimize chemotherapeutic entry (e.g.P-glycoprotein (Pgp) expression) or mechanisms to sequester weakly-basicdrugs in acidic organelles (e.g. recycling endosome, lysosome, andtrans-Golgi network).

Drug-loaded nanovectors, particularly the folate-targeted micelles, aretaken up by the DOX-resistant MCF-7 cells and rendered virtuallyinvisible to cancer cells, offering a potentially effective approach toovercoming multi-drug resistance. Studies using drug-loaded micelles mayalso answer questions regarding the time-dependant release from thezinc-crosslinked micelles. For example, in comparison with free DOX andCPT, drug release from the crosslinked micelle formulation may besustained over a much longer time period. Previously reported studies onuncrosslinked, DOX-loaded micelles suggest that similar therapeuticeffects are achieved with fewer doses and lower concentrations ofmicellar DOX.⁸⁴

Example 11 Cytotoxicity Analysis

While the block copolymer components and reagents used in these studiesare generally recognized as safe, experiments are conducted to determinetheir cytotoxic concentration limits, if any, in five cell lines: MCF-7,DOX-resistant MCF-7, HeLa, HepG2, and Chinese hamster ovary. Cellviability tests are performed using a highly sensitive, ATP-based assay(Celltiter-Glo™, Promega) which uses the luciferase reaction to measurethe number of viable cells in culture. The reagent is prepared by mixingwith an appropriate buffer and then added directly to multi-well platescontaining the cells to be tested. After mixing for two minutes, theplates are allowed to equilibrate at room temperature for 10 minutes andthen luminescence is measured using a luminometer equipped with a platereader. This particular method was chosen because it is homogeneous withonly a few plate-handling steps, data is collected within minutes afteradding the Celltiter-Glo™ reagent, and it is more sensitive thantraditional colormetric and fluorometric assays (e.g. MTT, alamarBlue,Calcein-AM). In addition to luminescence response due to apoptosis ornecrosis, one must consider other variables which may change the cellnumber to luminescence relationship using this particular assay. Theseinclude the use of multi-well plates suitable for luminescencemeasurements, ATP variations due to cell density, and adequate mixing toensure lysis and extraction of ATP from cells. Before cytotoxicitymeasurements are made, each cell line is seeded at ten differentdensities ranging from 0 to 50,000 cells per well, and the cellviability is tested using the cell viability assay described above.Since a linear relationship exists between luminescence (relativeluminescence units) and the number of cells in culture, potential assayproblems should be evident by large deviations from the standard, linearrelationship. Also, an ATP standard curve, prepared using multi-wellplates with varying concentrations of ATP in growth medium and recordingluminescence following addition of the Celltiter-Glo™ reagent, are alsouseful in identifying procedural errors in the assay. In general, atetrazolium salt MTT cell viability assay, which measures mitochondriafunction, are used in place of the ATP-based assay.

To ascertain the cytotoxicity of the nanovectors and crosslinkingreagents, viability studies comparing various concentrations of theblock copolymer micelles (without DOX or CPT) using the five cell linesdescribed previously are performed in triplicate using the previouslydetailed ATP-based assay. Specifically, each of the five cell lines aregrown to 80% confluence and seeded at 7000 cells per well and incubatedfor 1 day at 37° C. in a humidified atmosphere with 5% CO₂. Followingincubation, the culture media is replaced with an equal volume of mediacontaining 0 (control), 5, 10, 20, or 40 μg/mL concentration of themultiblock copolymer. It should be noted that all of theseconcentrations are above the critical micelle concentration(approximately 3 pg/mL), and therefore, the majority of the blockcopolymer is present in micellar form. Zinc-crosslinked micelles(without DOX or CPT) will also be evaluated in each of the cell lines atsimilar concentrations as described above. Assays are performed at 1, 3,and 5 days with no media change over the 5 day period, and cellviability is expressed as a percentage relative to samples grown withoutthe block copolymer (control).

Once the biocompatibility of the zinc-crosslinked and uncrosslinkedmulti-block copolymer micelles has been demonstrated, detailed cellviability studies are performed comparing free DOX and CPT versus theircrosslinked and uncrosslinked micellar analogues. MCF-7, DOX-resistantMCF-7, HeLa, HepG2, and Chinese hamster ovary cells are plated into a96-well plates (7,000 cells/well) and incubated in appropriate media for1 day. Free DOX and CPT are added to the media in concentrations of 0,0.001, 0.01, 0.1, 1, and 10 μM, and the cells are incubated for 1, 3,and 5 days without media change. Cell viability assays are performed intriplicate using the Celltiter-Glo™ reagent according the previouslydescribed protocol, and the data is averaged and plotted against drugconcentration as a best fit sigmoidal curve by using a nonlinearcurve-fitting algorithm. IC₅₀ values are reported as the drugconcentration resulting in 50% cell viability. These experiments arerepeated with uncrosslinked micelles, crosslinked micelles, and folate(in MCF-7 cells) or RGD-conjugated, crosslinked micelles (in HepG2cells). The quantity of multiblock copolymer is adjusted, based oncalculated micelle drug-loading values, to achieve comparable drugconcentrations of 0, 0.001, 0.01, 0.1, 1, and 10 μM. IC₅₀ values arecalculated for each sample set to determine which formulation is mostefficient in killing cancer cells. Cell viability is monitored over afive day period to ensure that adequate data points are obtained toproperly evaluate all micelle formulations. For example, after 24 hours,free DOX might demonstrate enhanced cytotoxicity when compared to acrosslinked, micellar formulation, but over longer time periods (i.e. 5days), the micelle formulation may prove to be more effective due toslow release from the micelle core.

Hemolysis studies comparing various concentrations of neutraldoxorubicin and camptothecin versus drug-loaded micelles, bothcrosslinked and uncrosslinked, are performed using red blood cells (RBC)isolated from whole human blood. Stock solutions containing isolated RBCin PBS (108 RBC per 200 μL) at pH values of 5.8, 6.6, 7.4 are prepared,and various concentrations of the drugs, polymer micelles, anddrug-loaded micelles are incubated at 37° C. for 1 hour in each of thethree RBC stock solutions. Following incubation, each RBC solution issubjected to centrifugation, and the hemoglobin release is determinedspectrophotometrically at 541 nm. Hemolytic activity of each sample atvarying pH values are expressed as a percentage of hemoglobin releaserelative to a 1% v/v Triton X-100 solution (positive control), which isassumed to give close to 100% hemolysis. Free DOX and CPT are evaluatedat concentrations of 0 (negative control), 50, 100, 200, and 400 μg/mLat each of the three pH values. Drug-loaded, multi-block copolymermicelles, both zinc-crosslinked and uncrosslinked, are assayed at pH5.8, 6.6, and 7.4 and compared to the hemolytic activity of the freedrug. The total polymer concentrations are adjusted, depending on knownmicelle drug loading, to achieve comparable DOX and CPT concentrationsof 0, 50, 100, 200, and 400 μg/mL. We anticipate that the hemolyticactivity of the micelle-encapsulated drug is dramatically reduced whencompared to the free drug. Previous hemolysis studies of DOX-loadedPEG-b-poly(caprolactone) (PCL) showed no hemolytic activity of DOX inmicelle form up to 225 μg/mL as compared to significant hemolysis (>10%)observed for the free DOX at similar concentrations.

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments that have been represented by way of example.

1.-37. (canceled)
 38. A drug-loaded micelle comprising a triblockcopolymer, wherein said micelle has a drug-loaded inner core, acrosslinked outer core, and a hydrophilic shell, wherein the multiblockcopolymer is of formula III:

wherein: n is 10-2500; m is 5-50; m′ is 5-50; L is a metal crosslinkedamino acid side-chain group; R^(y) is a hydrophobic or ionic, natural orunnatural amino acid side-chain group; R^(l) is—Z(CH₂CH₂Y)_(p)(CH₂)_(t)R³, wherein: Z is —O—; Y is —O—; p is 0-10; t is0-10; and R³ is optionally substituted aliphatic group; Q is a valencebond; R^(2a) is —NR⁴C(O)R⁴; and each R⁴ is independently hydrogen or anoptionally substituted aliphatic.
 39. The drug-loaded micelle accordingto claim 38, wherein the metal is zinc.
 40. The drug-loaded micelleaccording to claim 39, wherein the crosslinked amino acid is asparticacid or glutamic acid.
 41. The drug-loaded micelle according to claim39, wherein the amino acid is histidine.
 42. The drug-loaded micelleaccording to claim 38, wherein said micelle is a mixed micelle.
 43. Thedrug-loaded micelle according to claim 38, wherein R¹ is


44. The drug-loaded micelle according to claim 38, wherein R^(2a) isselected from the group consisting of:


45. The drug-loaded micelle according to claim 38, wherein R^(y) is ahydrophobic amino acid side-chain selected from a phenylalanineside-chain, alanine side-chain, benzyl or alkyl glutamate side chain, abenzyl or alkyl aspartate side chain, or leucine side-chain, andoptionally one or more of tyrosine side-chain, serine side-chain,threonine side-chain, glutamic acid, or aspartic acid such that theoverall poly(amino acid) block is hydrophobic.
 46. The drug-loadedmicelle according to claim 45, wherein R^(y) is a hydrophobic amino acidside-chain selected from a mixture of phenylalanine and tyrosine or amixture of leucine and tyrosine, such that the overall poly(amino acid)block is hydrophobic.
 47. The drug-loaded micelle according to claim 38,wherein: n is about 200 to about 300; m is 5-25; m′ is 10-50; and R^(2a)is —NHC(O)CH₃.
 48. The drug-loaded micelle according to claim 38,wherein the micelle encapsulates a hydrophobic chemotherapeutic drugselected from Abarelix, Aldesleukin, Aldesleukin, Alemtuzumab,Alitretinoin, Allopurinol, Altretamine, Amifostine, Anastrozole, Arsenictrioxide, Asparaginase, Azacitidine, BCG Live, Bevacuzimab, Avastin,Fluorouracil, Bexarotene, Bleomycin, Bortezomib, Busulfan, Calusterone,Capecitabine, Camptothecin, Carboplatin, Carmustine, Celecoxib,Cetuximab, Chlorambucil, Cisplatin, Cladribine, Clofarabine,Cyclophosphamide, Cytarabine, Dactinomycin, Darbepoetin alfa,Daunorubicin, Denileukin, Dexrazoxane, Docetaxel, Doxorubicin,Doxorubicin hydrochloride, Dromostanolone Propionate, Epirubicin,Epoetin alfa, Erlotinib, Estramustine, Etoposide Phosphate, Etoposide,Exemestane, Filgrastim, floxuridine fludarabine, Fulvestrant, Gefitinib,Gemcitabine, Gemtuzumab, Goserelin Acetate, Histrelin acetate,Hydroxyurea, Ibritumomab, Idarubicin, Ifosfamide, Imatinib Mesylate,Interferon alfa-2a, Interferon alfa-2b, Irinotecan, Lenalidomide,Letrozole, Leucovorin, Leuprolide Acetate, Levamisole, Lomustine,Megestrol Acetate, Melphalan, Mercaptopurine, 6-MP, Mesna, Methotrexate,Methoxsalen, Mitomycin C, Mitotane, Mitoxantrone, Nandrolone,Nelarabine, Nofetumomab, Oprelvekin, Oxaliplatin, Paclitaxel,Palifermin, Pamidronate, Pegademase, Pegaspargase, Pegfilgrastim,Pemetrexed Disodium, Pentostatin, Pipobroman, Plicamycin, PorfimerSodium, Procarbazine, Quinacrine, Rasburicase, Rituximab, Sargramostim,Sorafenib, Streptozocin, Sunitinib Maleate, Talc, Tamoxifen,Temozolomide, Teniposide, VM-26, Testolactone, Thioguanine, 6-TG,Thiotepa, Topotecan, Toremifene, Tositumomab, Trastuzumab, Tretinoin,ATRA, Uracil Mustard, Valrubicin, Vinblastine, Vincristine, Vinorelbine,Zoledronate, or Zoledronic acid.
 49. The drug-loaded micelle accordingto claim 48, wherein the hydrophobic chemotherapeutic drug isDoxorubicin or Doxorubicin hydrochloride.
 50. The drug-loaded micelleaccording to claim 48, wherein the hydrophobic chemotherapeutic drug isGemcitabine.
 51. The drug-loaded micelle according to claim 48, whereinthe hydrophobic chemotherapeutic drug is Letrozole.